Journal: Molecular psychiatry

Article Title: MeCP2-regulated miRNAs control early human neurogenesis through differential effects on ERK and AKT signaling

doi: 10.1038/mp.2017.86

Figure Lengend Snippet: ( a ) Immunofluorescence for neuronal marker MAP2 in WT2, RTT-WT2, and RTT-Mut2 three-week differentiated neurons. MeCP2 immunostaining using a C-terminus antibody shows no expression in RTT-Mut2 neurons. ( b ) Graphs showing mean ± SEM relative to control MAP2 and DCX mRNA levels in the same three control and RTT patient-derived three-week neuronal samples. DNM2 mRNA is also shown as an example of non-altered mRNA expression. Stars depict statistical significance based on two tailed one sample t -test (*p < 0.05, **p < 0.01). N: RTT-Mut2 = 8, RTT-WT2 = 5. ( c - d ) BrdU labeling and MeCP2 staining ( c ) and BrdU quantification ( d ) in WT2, RTT-WT2, and RTT-Mut2 early born three-week neurons. Stars depict statistical significance based on ANOVA with Neuman Keuls test (***p < 0.001). N: WT2 = 7, RTT-mut2 = 8, RTT-WT2 = 6. ( e ) Immunofluorescence for neuronal marker MAP2 in MeCP2 shRNA (shMeCP2) and control (shControl) three week neurons. MeCP2 immunostaining shows no expression in shMeCP2 neurons. ( f ) Quantification of the number of primary, secondary, and tertiary neurites per cell reveals significant reductions in shMeCP2 cells (**p < 0.01, ***p < 0.001). N: shControl = 13, shMeCP2 = 13. ( g ) Graph showing mean ± SEM MAP2 and DCX mRNA relative expression following in MeCP2-shRNA and shControl three-week neurons. DNM2 mRNA is also shown as a control. N: shControl = 10, shMeCP2 = 10. ( h ) Graph showing mean ± SEM average neurite length in shMeCP2 vs shControl neuronal cultures (***p < 0.001). N: shControl = 12, shMeCP2 = 13. ( i - j ) BrdU labeling and MeCP2 staining ( i ) and BrdU quantification ( j ) in MeCP2-shRNA and shControl three-week neurons. N: shControl = 4, shMeCP2 = 3. Stars in (f),(h),(j) depict statistical significance based on two-tailed Student’s t -test (**p < 0.01, ***p < 0.001), and in (g) based on two-tailed one sample t -test (***p < 0.001). 18S rRNA was used as a normalizer for all mRNA qRT-PCRs.

Article Snippet: The following primary antibodies were used: MAP2 (Encor Biotechnology 1:1000, CPCA-MAP2); DCX (Aves Labs, 1:200, DCX); PAX6 (Millipore; 1:200, ab2237); Pax6 (DSHB 1:100, PAX6); TBR2 (Abcam; 1:400, ab23345); TBR1 (Abcam 1:400, ab31940).

Techniques: Immunofluorescence, Marker, Immunostaining, Expressing, Control, Derivative Assay, Two Tailed Test, Labeling, Staining, shRNA

Journal: Molecular psychiatry

Article Title: MeCP2-regulated miRNAs control early human neurogenesis through differential effects on ERK and AKT signaling

doi: 10.1038/mp.2017.86

Figure Lengend Snippet: ( a ) Representative DAPI immunofluorescence reveals structural differences (i.e., expanded ventricular zones) in RTT-Mut2 vs. RTT-WT2 patient-derived organoids differentiated for 5 weeks. Scale bar = 500 μm. ( b ) Quantification of ventricular zones as a percentage of overall DAPI content reveals a significant increase in the percentage of ventricular zones in RTT-Mut2 organoids (n = 6 slices taken from 3 organoids (WT) and 8 slices taken from 4 organoids (Mut)); ****p < 0.0001, two-tailed Student’s t -test. ( c ) RTT-Mut2 organoids exhibit a reduction in mean ventricle wall thickness, defined as the distance between apical and basal surfaces of the ventricle. Graph shows mean ± SEM. (n = 60 ventricles measured across 6 slices taken from 3 organoids (WT) and 148 ventricles measured across 8 slices taken from 4 organoids (Mut); ***p < 0.001, Mann-Whitney test). ( d ) Assessment of the cumulative distribution of ventricle wall thickness reveals significant differences in RTT-Mut2 versus RTT-WT2 organoids. (n = 60 ventricles measured across 6 slices taken from 3 organoids (WT) and 148 ventricles measured across 8 slices taken from 4 organoids (Mut). ***p < 0.001, Kolmogorov-Smirnov test). ( e ) Representative immunostaining for PAX6 (early neural progenitors; green), TBR2 (intermediate neural progenitors; red), MAP2 (dendrites; magenta), TBR1 (early-born layer 6 cortical neurons; green), and Doublecortin (DCX) (immature neurons; magenta) in RTT-WT2 (top) and RTT-Mut2 (bottom) cerebral organoids. Scale bar = 500 μM. Immunostaining showing RTT-WT2 organoids expressing MeCP2 (red) and absence of MeCP2 protein in RTT-Mut2 organoids (bottom). Scale bar = 500 μm. ( f ) Representative thresholded images of the staining performed in (e) in RTT-WT2 (left) and RTT-Mut2 (right). Note the small percentage of TBR2- and TBR1-expressing cells in mutant organoids. Scale bar = 500 μM. ( g ) Quantification of the percentage of organoid expressing the aforementioned progenitor or neuronal marker, normalized to DAPI revealed: 1) significant reduction in DCX in RTT-Mut2 organoids (n = 25 sections from a total of 11 organoids (WT) and 35 sections from a total of 14 organoids (Mut), distributed across three independent organoid differentiation batches); 2) significant decrease in the expression of MAP2 in RTT-Mut2 organoids (n = 26 sections from a total of 11 organoids (WT) and 33 sections from a total of 14 organoids (Mut), distributed across three independent organoid differentiation batches); 3) significant increase in the expression of PAX6 in RTT-Mut2 organoids (n = 26 sections from a total of 11 organoids (WT) and 35 sections from a total of 14 organoids (Mut), distributed across three independent organoid differentiation batches); 4) significant decrease in the expression of Tbr2 in RTT-Mut2 organoids (n = 27 sections from a total of 12 organoids (WT) and 36 sections from a total of 14 organoids (Mut), distributed across three independent organoid differentiation batches); and 5) a significant decrease in the expression of Tbr1 in RTT-Mut2 (n = 26 sections from a total of 11 organoids (WT) and 34 sections from a total of 14 organoids (Mut), distributed across three independent organoid differentiation batches). A total of 79 sections (WT) and 103 sections (Mut) were analyzed for this experiment, which were generated from 3 independent differentiation rounds of both RTT-WT2 and RTT-Mut2 organoids. (***p < 0.001, ****p< 0.0001, two-tailed Student’s t -test). ( h ) Mean ± SEM relative to RTT-WT2 miR-199 and miR-214 (normalized to RNU44), pri-miR-199-a1,-a2,-b, pri-miR-214, and BMP4 mRNA levels (normalized to 18S rRNA) in RTT-WT2 (green bars) and RTT-Mut2 (red bars) 5 week 3D cerebral organoids. Stars depict statistical significance based on two-tailed Student’s t -test (*p < 0.05, ***p < 0.001, ****p < 0.0001). N for miR-199/214: RTT-Mut2 = 10, RTT-WT2 = 6, N for pri-miRNAs/BMP4: RTT-Mut2 = 4, RTT-WT2 = 5. ( i ) Mean ± SEM relative to RTT-WT2 DCX, PAX6, GAD1, DLX1, SST, and PVALB mRNA levels (normalized to 18S rRNA) in RTT-WT2 (green bars) and RTT-Mut2 (red bars) 5 week 3D cerebral organoids. Stars depict statistical significance based on two-tailed Student’s t -test (**p < 0.01, ****p< 0.0001). N: RTT-Mut2 = 4, RTT-WT2 = 5. ( j ) Human iPSC-derived cell organoids co-electroporated with GFP and control vector or MeCP2 shRNAs and examined after 7 days. MeCP2 shRNA-targeted cells exhibit increased number of PAX6+ progenitors. Scale: 100 μm. High-magnification of cells are shown in ( k ) and ( l ). The asterisks in (k) denote the PAX6- cells in the control group. Increased PAX6+ progenitors after depletion of MeCP2 are denoted by arrows in (l). ( m ) The percentages of PAX6+ GFP+ cells were quantified. *p < 0.05 versus control; two-tailed Student’s t -test. More than 100 GFP+ neurons from three organoids were examined in each group. Bars in all graphs represent mean ± S.E.M. ( n-p ) RTT-WT2 and RTT-Mut2 organoids were electroporated with GFP and fluorescence beads were used to mark the ventricles. Electroporated GFP+ cells in RTT-Mut2 organoids exhibited reduced migration distance ( o ) as compared to cells in RTT-WT2 organoids ( n ). Scale: 100 μm. ( p ) Graph showing significant reduction of migration distance in electroporated GFP+ cells in RTT-Mut2 vs RTT-WT2 organoids. *p < 0.05 versus control; ***p < 0.001 versus control; two-tailed Student’s t -test. More than 700 GFP+ cells from seven organoids were examined in each group. Segments such as those selected in ( n,o ) were divided in 9 bins. Bars in all graphs represent mean ± S.E.M.

Article Snippet: The following primary antibodies were used: MAP2 (Encor Biotechnology 1:1000, CPCA-MAP2); DCX (Aves Labs, 1:200, DCX); PAX6 (Millipore; 1:200, ab2237); Pax6 (DSHB 1:100, PAX6); TBR2 (Abcam; 1:400, ab23345); TBR1 (Abcam 1:400, ab31940).

Techniques: Immunofluorescence, Derivative Assay, Two Tailed Test, MANN-WHITNEY, Immunostaining, Expressing, Staining, Mutagenesis, Marker, Generated, Control, Plasmid Preparation, shRNA, Fluorescence, Migration

Journal: Molecular psychiatry

Article Title: MeCP2-regulated miRNAs control early human neurogenesis through differential effects on ERK and AKT signaling

doi: 10.1038/mp.2017.86

Figure Lengend Snippet: ( a ) Representative Western blots showing levels of phosphorylated (p) and total (t) ERK1/2 and AKT, PTEN and PAK4 protein levels together with normalizer β-actin in MeCP2 (shMeCP2) or Control shRNA (shControl)- expressing WT NPs (left), as well as in WT2, RTT-Mut2 and RTT-WT2 NPs. ( b-c ) Graphs based on Western blot analysis showing mean ± SEM for the same proteins mentioned above in shMeCP2 (red bar) and shControl (blue bar) (b) and WT unaffected control (WT2 – blue bar), RTT-Mut2 (red bar) and RTT-WT2 (green bar) (c) samples. Stars depict statistical significance based on two-tailed one sample t -test (shMeCP2, shControl) or ANOVA (WT2, RTT-Mut2, RTT-WT2) with Dunnett’s multiple comparisons test (right) (*p < 0.05, **p < 0.01, ***p < 0.001). N: shControl = 4, shMeCP2 = 4, WT2 = 6, RTT-mut2 = 10, RTT-WT2 = 6. ( d , e ) Correlation between PAK4 and p/t ERK1/2 ( d ) and PTEN and p/t AKT levels ( e ) in WT2, RTT-WT2, RTT-Mut2, as well as shControl and shMeCP2 NPs (log transformed). Each dot represents one sample (Blue dots for positive and red for negative correlations). Spearmann coefficients and p-values are shown in the graphs. ( f ) Schematic of the proposed molecular mechanism. ( g ) Representative Western blots showing levels of phosphorylated and total ERK1/2 (pERK1/2 and tERK1/2) and AKT (pAKT and tAKT), and MeCP2 protein levels together with normalizer β-actin, following nucleofection of miR-199 or miR-214 inhibitors (anti-miR-199 and anti-miR-214) in shMeCP2 expressing NPs, as well as with shMeCP2 and shControl NPs nucleofected with a negative miRNA control inhibitor (shMeCP2 anti-NC and shControl anti-NC respectively). ( h ) Graphs showing mean ± SEM PAK4 mRNA (upper) and phosphorylated vs total ERK1/2 protein in shControl anti-NC, shMeCP2 anti-NC, shMeCP2 anti-miR-199, and shMeCP2 anti-miR-214 NPs. ( i ) Graphs showing mean ± SEM PTEN mRNA (upper) and phosphorylated vs total AKT protein in the NP samples as above. For all 4 groups N = 5 for protein and N = 8 for mRNA measurements. ( j ) Representative immunostaining for MAP2 and MeCP2 in three-week anti-miRNA nucleofected MeCP2 shRNA neurons (same groups as above- yet different developmental stage). ( k ) Graphs showing mean ± SEM DCX (upper) and MAP2 mRNA in the three-week neuronal samples as above. N = 8 for for all 4 groups. Analysis in (h), (i), (k) was based on two-tailed one sample t -test, (*p < 0.05,**p < 0.01, # 0.10< p < 0.05). 18S rRNA was used as a normalizer for all mRNA qRT-PCRs.

Article Snippet: The following primary antibodies were used: MAP2 (Encor Biotechnology 1:1000, CPCA-MAP2); DCX (Aves Labs, 1:200, DCX); PAX6 (Millipore; 1:200, ab2237); Pax6 (DSHB 1:100, PAX6); TBR2 (Abcam; 1:400, ab23345); TBR1 (Abcam 1:400, ab31940).

Techniques: Western Blot, Control, shRNA, Expressing, Two Tailed Test, Transformation Assay, Immunostaining