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

Journal: Hippocampus

Article Title: P2X7 expression patterns in the developing Fmr1-knockout mouse hippocampus.

doi: 10.1002/hipo.23634

Figure Lengend Snippet: FIGURE 2 P2X7 equally colocalized with dentate gyrus (DG) hilus MASH1+ stem cells and doublecortin (DCX)+ neuroblasts in wild-type (WT) and knockout (KO) mice at P14. Hippocampus sagittal sections were probed with antibodies targeting P2X7 and MASH1 (a) or P2X7 and DCX (c) to identify colocalization with Type 2a progenitors and Type 3 neuroblasts, respectively. Percentage (%) of P2X7 colocalization within the DG hilus was calculated by overlaying threshold binaries of P2X7 and MASH1 (b) or DCX (d). Error bars show mean ± SEM. Scale bars, 50 μm.

Article Snippet: The primary antibodies included the following: P2X7 (rabbit polyclonal; dilutions ranged from 1:100 to 1:500 and all comparisons were made with consistent dilutions; Alomone Labs; CAT no.: APR-004), MASH1 (goat polyclonal; 1:500; R&D Systems; CAT no.: AF2567), doublecortin (DCX; goat polyclonal; 1:500; Novus Biologicals; CAT no.: NBP1-72042), NeuN (mouse monoclonal; 1:500; Abcam; CAT no.: ab104224), and glial fibrillary acidic protein (GFAP; chicken polyclonal; 1:500; Invitrogen; CAT no.: PA1-10004).

Techniques: Knock-Out

Journal: Scientific reports

Article Title: Establishment of a high-content compatible platform to assess effects of monocyte-derived factors on neural stem cell proliferation and differentiation.

doi: 10.1038/s41598-024-57066-2

Figure Lengend Snippet: Figure 2. MoC-induced NSC proliferation and differentiation is dependent on monocytic and neural stem cell microenvironment. (A) Representative images of KI67 (Orange), A2B5 (Green) and DCX (Red) after differentiation. White bar is 50 µm long. (B) NSC differentiation as measured by the differentiation markers A2B5 and DCX as well as the proliferation marker KI67. Three different conditioned media (listed at the top) were tested in combination with three different basal media (listed on the right). Each conditioned medium was added at 5%, 12.5% or 25% (percentage shown on x axis). Each dot represents the average number of positive cells in one well (384 well plate). Three independent experiments were performed, each with 16 wells per condition. Results were normalized (z-score normalization) and analyzed together, using mixed linear models (results summarized in Table 2).

Article Snippet: Primary antibodies including A2B5 (R&D Systems, MAB1416), DCX (Novus Biological, NBP1-72042), KI67 (Abcam, Ab16667), NG2 (BD 554275), OLIG2 (Millipore, AB9610), SOX10 (R&D Systems, AF2864), GALC (Merck, MAB342), PDGFRɑ (CST, 5241T), O4 (R&D Systems, MAB1326), NEUN (CST, 24307), PLP (Sigma Aldrich, MAB388), MBP (Sigma Aldrich, AB9348), βIIITUB (Biolegend, 801213), GFAP (Agilent, Z0334), CASPASE3 (R&D Systems, MAB835), PCNA (Santa Cruz Biotechnology, sc-56), NESTIN (R&D Systems, MAB1259), SOX1 (R&D Systems, AF3369) and SOX2 (R&D Systems, AF2018) were incubated in blocking solution at 4 °C overnight.

Techniques: Marker

Journal: Scientific reports

Article Title: Establishment of a high-content compatible platform to assess effects of monocyte-derived factors on neural stem cell proliferation and differentiation.

doi: 10.1038/s41598-024-57066-2

Figure Lengend Snippet: Figure 3. Passage-dependent intrinsic properties affect self-renewal and differentiation capacity of NSCs. (A) Representative images of a neurosphere formation assay with low, middle and high passage cells. High passage cells do not form neurospheres. (B) Quantification of neurosphere formation assays. Neurospheres from low passage cells do not differ from neurospheres formed by middle passage cells in their quantity or size (top and bottom panels respectively). Data described by summary statistics including mean and SD for neurosphere count and violin plot for neurosphere diameter (top and bottom panels respectively). Each dot represents one image. Neurosphere formation assay was performed three times, each time ten random images were taken (n = 3). (C) Quantification of differentiation markers DCX and NG2 after differentiation with low, middle and high passage cells. With increasing passage number cells differentiate more likely into the glial lineage while decreasing their differentiation into the neuronal lineage. Each dot represents one well (96 well plates); two independent experiments with 10 wells per passage were performed, standardized (z-score standardization) and plotted together. Statistics were performed for each individual experiment and largest p-values are displayed. Kruskal Wallis one way ANOVA, followed by Tukey’s HSD post-hoc test. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Primary antibodies including A2B5 (R&D Systems, MAB1416), DCX (Novus Biological, NBP1-72042), KI67 (Abcam, Ab16667), NG2 (BD 554275), OLIG2 (Millipore, AB9610), SOX10 (R&D Systems, AF2864), GALC (Merck, MAB342), PDGFRɑ (CST, 5241T), O4 (R&D Systems, MAB1326), NEUN (CST, 24307), PLP (Sigma Aldrich, MAB388), MBP (Sigma Aldrich, AB9348), βIIITUB (Biolegend, 801213), GFAP (Agilent, Z0334), CASPASE3 (R&D Systems, MAB835), PCNA (Santa Cruz Biotechnology, sc-56), NESTIN (R&D Systems, MAB1259), SOX1 (R&D Systems, AF3369) and SOX2 (R&D Systems, AF2018) were incubated in blocking solution at 4 °C overnight.

Techniques: Tube Formation Assay

Journal: Scientific reports

Article Title: Establishment of a high-content compatible platform to assess effects of monocyte-derived factors on neural stem cell proliferation and differentiation.

doi: 10.1038/s41598-024-57066-2

Figure Lengend Snippet: Figure 4. NSC-intrinsic properties influence susceptibility to monocyte/MoC-mediated NSC proliferation and differentiation. Quantification of KI67, A2B5 and DCX after differentiation in monocyte conditioned media. OPC (top row) and SP (bottom row) conditioned media were tested in low (black) and middle (gray) passage NSCs. Conditioned medium was added at 25%. Each dot represents the average number of positive cells in one well (384 well plate). Experiment was performed including n = 5 healthy donors, each with 3 wells (technical replicates) per condition. Results were normalized (z-score normalization) and analyzed together, using mixed linear models (results summarized in Table 2).

Article Snippet: Primary antibodies including A2B5 (R&D Systems, MAB1416), DCX (Novus Biological, NBP1-72042), KI67 (Abcam, Ab16667), NG2 (BD 554275), OLIG2 (Millipore, AB9610), SOX10 (R&D Systems, AF2864), GALC (Merck, MAB342), PDGFRɑ (CST, 5241T), O4 (R&D Systems, MAB1326), NEUN (CST, 24307), PLP (Sigma Aldrich, MAB388), MBP (Sigma Aldrich, AB9348), βIIITUB (Biolegend, 801213), GFAP (Agilent, Z0334), CASPASE3 (R&D Systems, MAB835), PCNA (Santa Cruz Biotechnology, sc-56), NESTIN (R&D Systems, MAB1259), SOX1 (R&D Systems, AF3369) and SOX2 (R&D Systems, AF2018) were incubated in blocking solution at 4 °C overnight.

Techniques: