Journal: bioRxiv

Article Title: Dynamic Regulation OF The Chromatin Environment By Ash1L Modulates Human Neuronal Structure And Function

doi: 10.1101/2024.12.02.625500

Figure Lengend Snippet: (A) Diagram of ASH1L protein domains showing the location of the pathogenic variant E2148* (blue) in ASH1L catalytic domain and itsr associated clinical phenotypes. (B) Illustration depicts the dual SMAD inhibition protocol used to generate cortical excitatory human neurons. ( C ) ASH1L expression was quantified by qPCR using human neurons at day 35 of neuronal induction. Fold change is normalized to control. Bar represents the mean and individual measures from four independent experiments are shown for control (grey with open circles), and E2148* (light blue with solid blue circles). Samples were analyzed as a ratio of the control. Statistical analysis was conducted using unpaired t-test. **** P < 0.0001. ( D ) Representative images are shown for human neurons from control, and E2148* cultures at day 35 of neuronal induction. Neurons stained with MAP2 are shown in black and white for ease of viewing. Calibration bars represent 20µm. ( E-H ) Morphogenesis measures are shown for four independent experiments for control neurons (grey bar with open circles), and E2148* mutant neurons (light blue bars with solid dark blue circles). Individual points represent the average of 4 independent experiments, an average of 30 neurons were measured per experiment. ( E ) Mean neurite length is shown for control (n=124 neurons; 56.9 ± 2.41), and E2148* (n=118 neurons; 47.47 ± 1.99). Grouped statistical analysis was conducted using unpaired t-test, **P < 0.004. ( F ) Total neurite length is shown for control (n=124 neurons; 182.7 ± 6.39), and E2148* (n=118 neurons; 139.3 ± 4.66). Grouped statistical analysis was conducted using unpaired t-test, **** P < 0.0001. ( G ) Neuronal morphology analyzed by measuring the complexity index (see methods). Calculations were conducted after identifying outliers using the ROUT 1% method for control (n=115; 289.5 ± 18.21), and E2148* (n=112; 228.8 ± 13.42). Grouped statistical analysis was conducted unpaired t-test ** P < 0.0099. ( H ) Cell soma size was analyzed for three independent experiments by measuring the area for control (n=96; 77.67 ± 3.47), and E2148* (n=91; 69.15 ± 2.51). Statistical analysis was conducted using unpaired t-test P=0.056. ( I ) Sholl analysis was used to measure neuronal arborization. The number intersections away from the cell soma were measured every 10µm and are shown for control (open gray circles), and E2148* (solid dark blue circles) neurons from 10µm to 120µm. Statistical analysis was conducted using a mixed model effects *** P < 0.0006, and **** P < 0.0001. ( J-L ) Analysis of H3K36me2 and H3K4me3 levels on chromatin fraction for four independent experiments is shown for neurons at day 41 of neuronal induction. ( J ) Representative western blot shows H3K36me2, H3K4me3 and histone H3 for control, and E2148* neurons. H3 Histone marks were normalized to histone H3 levels for analysis. ( K ) H3K36me2 protein levels are shown for control (1± 0), and E2148* (0.67 ± 0.26). ( L ) H3K4me3 protein levels are shown for control (1± 0), and E2148* (0.68± 0.11). (K -L ) Statistical analysis was conducted using unpaired t-test *P< 0.025. Not significant P value is not shown.

Article Snippet: Primary antibody for MAP2 (Aves lab, #MAP) was added and cells were incubated overnight at 4°C, followed by secondary antibody incubation for 1 hour at room temperature using Alexa Fluor 647 (Thermo Scientific, #A-21449) at a dilution of 1:1000.

Techniques: Variant Assay, Inhibition, Expressing, Control, Staining, Mutagenesis, Western Blot

Journal: bioRxiv

Article Title: Dynamic Regulation OF The Chromatin Environment By Ash1L Modulates Human Neuronal Structure And Function

doi: 10.1101/2024.12.02.625500

Figure Lengend Snippet: ( A ) PCA plots shows biological replicates (n=4) for control (green), and E2148* (salmon) neurons RNA seq experiments. ( B ) Heatmap shows top 100 DEGs for control (green), and E2148* (salmon) neurons at day 35 (n=4 biological replicates). The top 15 DEGs are listed. ( C ) Volcano plots showing DEGs in the heterozygous E2148* mutant iPSC-derived neurons. Log 2 fold changes (LFC) gene expression (x-axis) and -log 10 adjusted P values (y-axis) generated from DESeq2 differential gene expression analysis are shown. Vertical dotted lines represent 0.58 LFC (1.5 FC) and horizontal dotted line shows adjusted P=0.05. Significant DEGs are shown in red with the top 20 labelled in the plot. ( D-F ) Functional enrichment analysis by EnrichR for biological process ( D ), cellular compartment ( E ), and molecular function ( F ) show enrichment for all DEGs, upregulated and downregulated DEGs in E2148* mutant neurons vs. control neurons. Circle size represents the number of DEGs in that category and the color represents the adjusted P value. ( G ) Correlation of gene length to fold change analyzed for all significant DEGs in E2148* (blue line) mutant neurons. Grey shade shows the variability across samples. ( H ) Analysis of gene length in upregulated (blue) and downregulated (red) DEGs for E2148* neurons. ( I ) Analysis of de novo transcription by EU click chemistry at day 41 of neuronal differentiation. Representative images of human neurons that incorporated EU (gray), stained with neuronal marker MAP2 (cyan) and nuclear marker DAPI (blue) are shown for control (top row), and E2148* (bottom row). Enlarged nuclei stained with EU is shown. Calibration bars are 20µm. ( J-K ) Measurements of EU incorporation are shown for control neurons (grey bars with open circles), and E2148* (light blue bars with solid deep blue circles) mutant neurons. Mean and standard error are shown with individual dots representing the average of individual measures for five independent experiments. ( I ) Pearsons’ correlation coefficient analysis is shown for five independent experiments for control (n=187; 0.785 ± 0.003), and E2148* (n=115; 0.746 ± 0.005) neurons. ( J ) EU nuclear intensity normalized to control is shown for five independent experiments for control (n=187; 1.017 ± 0.029), and E2148* (n=115; 0.817 ± 0.027) neurons. ( I-J ) Grouped data analyzed using unpaired t test with Welch’s correction, ****P < 0.0001. Not significant P values are not shown.

Article Snippet: Primary antibody for MAP2 (Aves lab, #MAP) was added and cells were incubated overnight at 4°C, followed by secondary antibody incubation for 1 hour at room temperature using Alexa Fluor 647 (Thermo Scientific, #A-21449) at a dilution of 1:1000.

Techniques: Control, RNA Sequencing, Mutagenesis, Derivative Assay, Gene Expression, Generated, Functional Assay, Staining, Marker

Journal: bioRxiv

Article Title: Dynamic Regulation OF The Chromatin Environment By Ash1L Modulates Human Neuronal Structure And Function

doi: 10.1101/2024.12.02.625500

Figure Lengend Snippet: (A) Representative images are shown for day 35 human neurons from control, and E2148* cultures treated for 3 days with DMSO, Tazemetostat (0.5µM) and Vorinostat (0.1µM). Neurons stained with MAP2 are shown in black and white for ease of viewing. Calibration bars represent 30µm. ( B-F ) Morphogenesis analysis is shown for at least 4 independent experiments (unless otherwise annotated) in which we measured at least 30 neurons per experiment for control (grey bar with open circles) and E2148* (light blue bars with solid dark blue circles) neurons treated with either DMSO, Tazemetostat (TAZ) or Vorinostat (VOR). Individual points represent the average of multiple independent experiments. ( B ) Total neurite length is shown as the mean (bar) with the average of individual measurements represented by the circles for: control + DMSO (n=93 neurons; 231.9 ± 7.27); control + TAZ (n=119; 184.4± 5.46); control + VOR (n=118 neurons; 227.3± 6.8); E2148* + DMSO (n=113 neurons; 163.3 ± 4.83); E2148* + TAZ (n=112; 200.5± 5.94); E2148* + VOR (n=129 neurons; 225.2± 8.38). ( C ) Mean neurite length is shown as the mean (bar) with the average of individual measurements represented by the circles for: control + DMSO (n=90 neurons; 69.86 ± 2.409); control + TAZ (n=117; 64.16± 2.04); control + VOR (n=118 neurons; 81.28± 2.75); E2148* + DMSO (n=112 neurons; 55.75 ± 1.88); E2148* + TAZ (n=111; 66.16 ± 2.47); E2148* + VOR (n=126 neurons; 76.02 ± 2.58). ( D ) Complexity index measurements were first analyzed using the “identify outliers” ROUT function in graph pad and are shown as the mean (bar) with the average of individual measurements represented by the circles for: control + DMSO (n=90 neurons; 390± 26.88); control + TAZ (n=111; 329.4 ± 22.40); control + VOR (n=116 neurons; 461.8 ± 25.45); E2148* + DMSO (n=116 neurons; 261.9 ± 18.80); E2148* + TAZ (n=105; 357.6 ± 20.83); E2148* + VOR (n=125 neurons; 458.3 ± 29.66). ( B-D ) Statistical analysis of grouped measurements was conducted using TWO-way ANOVA with Tukey’s test for multiple comparisons: * P < 0.04 ** P < 0.009, *** P < 0.0006, **** P < 0.0001. ( E ) Sholl analysis was used to measure neuronal arborization across three different treatments in the E2148* mutant neurons. The number intersections away from the cell soma were measured every 10µm and are shown for E2148* + DMSO (inverted dark blue triangles), E2148* + TAZ (open triangles), and E2148* + VOR (solid light blue triangles) neurons. Statistical analysis by TWO-way ANOVA with mixed model effects * P < 0.05, ** P < 0.009, and *** P = 0.0008. Green asterisk (E2148* +DMSO vs. E2148* + VOR), red asterisk (E2148* + DMSO vs. E2148* + TAZ). ( F ) Sholl analysis is shown to compare the most effective treatment (vorinostat) to the untreated control and E2148* mutant neurons. The number of intersections away from the cell soma were measured every 10µm and are shown for control+ DMSO (open gray circles), E2148* + DMSO (solid dark blue circles) and E2148* + VOR (half pink/light blue circles) neurons. Statistical analysis by TWO-way ANOVA with mixed model effects * P < 0.05, ** P < 0.005, *** P = 0.0005, and **** P < 0.0001. Green asterisk (E2148*+DMSO vs. Control + DMSO), red asterisk (E2148* + DMSO vs. E2148* + VOR). ( G-K ) Analysis of nuclear levels of H3K27me3 and H4K16ac in four independent experiments (unless otherwise indicated) across all treatments is shown for neurons at day 35 of neuronal induction. ( G ) Representative images of nuclear H3K27me3 (red) are shown for either DMSO (left column) or Tazemetostat (right column) treated control, or E2148* mutant neurons stained with MAP2 (cyan) and nuclei is stain with DAPI (blue). ( H ) Quantification of H3K27me3 nuclear levels measured by mean gray value is shown for all treatments. Measurements from at least 3 independent experiments with at least 30 neurons analyzed per experiment were analyzed as a group and are shown as the mean (bar) with the average of individual measurements represented by the circles for: control + DMSO (n=134 neurons; 547.4 ± 13.19); control + TAZ (n=154; 258.1 ± 10.83); control + VOR (n=101 neurons; 545.5 ± 19.07); E2148* + DMSO (n=141 neurons; 471.0 ± 10.87); E2148* + TAZ (n=140; 301.1 ± 8.03); E2148* + VOR (n=103 neurons; 539.4 ± 18.79). ( I ) Representative images of nuclear H4K16ac (red) are shown for either DMSO (left column) or Tazemetostat (right column) treated control, and E2148* mutant neurons stained with MAP2 (cyan) and nuclei is stain with DAPI (blue). ( J ) Quantification of H4K16ac nuclear levels measured by mean gray value is shown for all treatments. Measurements from at least 3 independent experiments with at least 30 neurons analyzed per experiment were analyzed as a group and are shown as the mean (bar) with the average of individual measurements represented by the circles for: control + DMSO (N= 4 experiments; n=114 neurons; 322.8 ± 12.85); control + TAZ (n=95; 360.5 ± 13.04); control + VOR (n=145 neurons; 581.6 ± 16.45); E2148* + DMSO (n=166 neurons; 270.3 ± 10.33); E2148* + TAZ (n=106; 350.4 ± 11.81); E2148* + VOR (n=158 neurons; 654.7 ± 9.49). ( H and J ) Statistical analysis of grouped measurements was conducted using TWO-way ANOVA with Tukey’s test for multiple comparisons: * P < 0.05, *** P < 0.005, *** P < 0.0005, **** P < 0.0001.

Article Snippet: Primary antibody for MAP2 (Aves lab, #MAP) was added and cells were incubated overnight at 4°C, followed by secondary antibody incubation for 1 hour at room temperature using Alexa Fluor 647 (Thermo Scientific, #A-21449) at a dilution of 1:1000.

Techniques: Control, Staining, Mutagenesis

Journal: Biology

Article Title: In Vitro Modeling as a Tool for Testing Therapeutics for Spinal Muscular Atrophy and IGHMBP2-Related Disorders.

doi: 10.3390/biology12060867

Figure Lengend Snippet: Figure 1. Direct conversion of skin-derived fibroblasts into induced neurons (iNs): (A) Schematic of the direct conversion protocol. Neurons (iNs) were generated in 7 days using seven small molecules: VPA, CHIR99021, Repsox, Forskolin, SP600125, GO6983 and Y-27632. (B) Fluorescent microscope imaging at 20× magnification shows directly converted iNs from a healthy line. iNs were checked for expression of pan-neuronal markers such as Tuj1 (green) and Map2 (red) (top row), as well as inhibitory neuronal marker GABA (green) and glutamatergic neuronal marker vGLUT1 (red) (middle row). The neuronal mixed population does not contain cholinergic neurons (lower row). Scale bar = 100 µm. HFM: human fibroblast medium, NMM: neuron maturation medium.

Article Snippet: Primary antibodies against TUJ1 (Biolegend, San Diego, CA, USA), Map2 (Novus Biologics, Centennial, CO, USA), GABA (Novus Biologics) and vGLUT (Thermo Fisher, Waltham, MA, USA) were diluted in blocking solution.

Techniques: Derivative Assay, Generated, Microscopy, Imaging, Expressing, Marker

Journal: Scientific Reports

Article Title: Neuronal morphology and synaptic input patterns of neurons in the intermediate nucleus of the lateral lemniscus of gerbils

doi: 10.1038/s41598-023-41180-8

Figure Lengend Snippet: Primary antibodies used for histology.

Article Snippet: MAP2 , Chicken , Polyclonal , AMCA , 1:1000 , OriGene , TA336617.

Techniques:

Journal: Scientific Reports

Article Title: Neuronal morphology and synaptic input patterns of neurons in the intermediate nucleus of the lateral lemniscus of gerbils

doi: 10.1038/s41598-023-41180-8

Figure Lengend Snippet: Position and transmitter content of nuclei in the lateral lemniscus. ( a ) Nissl staining of the lateral lemnisicus and inferior colliculus. Black circle indicates position of the DNLL, magenta circle the position of the INLL and pink square the position of the VNLL. Scale bar equals 1 mm. ( b ) GABA (green) and glycine (red) immunofluorescence in the DNLL, INLL and VNLL (from left to right). Scale bare equals 100 µm. ( c ) Co-labelling of MAP2 (blue), GABA (green) and glycine (red) immunofluorescence in the DNLL, INLL, VNLL and MNTB as indicated. Triple staining is given on the left, the single fluorescence of each channel is given in black in the indicated order: MAP-2, GABA and glycine. Scale bar equals 50 µm.

Article Snippet: MAP2 , Chicken , Polyclonal , AMCA , 1:1000 , OriGene , TA336617.

Techniques: Staining, Immunofluorescence, Fluorescence

Journal: PLoS ONE

Article Title: Ectopic Expression of Neurogenin 2 Alone is Sufficient to Induce Differentiation of Embryonic Stem Cells into Mature Neurons

doi: 10.1371/journal.pone.0038651

Figure Lengend Snippet: (A-D) Wide field scans of Tuj1 staining 5 days post transfection (dpt). Dark field view (A, B) showing Tuj1 positive cells in Ngn2 -transfected cells (A), but not in mock-transfected cells (B). Corresponding phase contrast images (C,D) reveal the presence of stem cell like colonies (arrows). Scale bars: 200 µm. (E-H) Close-up views of developing neurons 5dpt expressing Tuj1 (E,F) and Map2ab (G,H). Scale bars: 20 µm. (I-N) Loss of Nanog expression (arrowheads) 3dpt in Ngn2 -transfected (I,K,M), but not in mock-transfected cells (J,L,N). Transfected cells are visualized by expression of cotransfected GFP (I,J). Scale bars: 20 µm. (O) Gene expression pattern of untreated (ut), Ngn2 -transfected (d5+,d7+), and mock-transfected (d5-,d7-) mESCs 5 and 7dpt. b: Brain cDNA. Dashed line indicates grouping of different parts from the same gel. A representative result from three independent experiments is shown. (P) Tuj1 positive cells in Ngn2 -transfected and mock-transfected cells 5 and 7dpt. Absolute numbers are shown as non-differentiating cells continue proliferating. Therefore, the relative number would not really reflect the increase of neurons upon Ngn2 compared to mock transfection. Mean numbers +/− SD of three independent experiments are shown.

Article Snippet: Immunofluorescence staining was performed as described using anti-Tuj1 antibody (Novus Biologicals, 1∶1000), anti MAP2ab antibody (Acris Antibodies GmbH, 1∶250), anti-Nanog-antibody (antikoerper-online, 1∶1000), anti-Stat3 antibody (Santa Cruz, 1∶1000), anti-vGlut1 antibody (SynapticSystems, 1∶1000), anti-tau antibody (SynapticSystems, 1∶1000), anti-NR1 antibody (Sigma, 1∶1000), anti-Synapsin 1 (SynapticSystems, 1∶1000), anti-MAP2 (Chemicon, 1∶500), anti-Tuj1 (R&D systems, 1∶600), anti-th (Sigma, 1∶500), anti-myc (cell signalling, 1∶2000), Hoechst 33258 (Molecular probes), and DAPI (Sigma).

Techniques: Staining, Transfection, Expressing, Gene Expression