Journal: bioRxiv

Article Title: Nervous system reduction in branched-chain amino acid metabolism disrupts hippocampal neurogenesis and memory

doi: 10.1101/2022.06.10.495711

Figure Lengend Snippet: A) Representative wide field and higher magnification images from 6-month-old control and Ppm1k cKO mouse hippocampal sections stained for doublecortin (DCX) in green and DAPI in blue. Below: Quantification of the number of DCX+ cells found in the GCL of control and Ppm1k cKO mice per 0.1 mM of coronal tissue of the dentate gyrus. Ctrl n = 6, cKO n = 6; ***p < 0.001. Quantification of the number of DCX+ cells found in the SGZ of control and Ppm1k cKO mice per 0.1 mM of coronal tissue of the dentate gyrus. Ctrl n = 6, cKO n = 6; *p < 0.05. Subgranular zone (SGZ), granular cell layer (GCL), molecular layer (ML). Scale bars = 150 μm, and 40 μm. B) Representative images from 6-month-old control and Ppm1k cKO mouse coronal hippocampal sections stained for DCX in green and NeuN in red. Below: Quantification of the number of cells positive for both DCX and NeuN in the GCL of control and Ppm1k cKO mice per 0.1 mM of coronal tissue of the dentate gyrus. Ctrl n = 6, cKO n = 6; ***p < 0.001. C) Representative images from 6-month-old control and Ppm1k cKO mouse coronal hippocampal sections stained DCX in green and NeuroD1 in red. Below: Quantification of the number of cells positive for NeuroD1 in the GCL of control and Ppm1k cKO mice per 0.04 mM of coronal tissue of the dentate gyrus. Ctrl n = 3, cKO n = 3; ***p < 0.001. D) Representative images from 6-month-old control and Ppm1k cKO mouse coronal hippocampal sections stained for GFAP in red, Edu in green, and DAPI in blue. Right: Quantification of the number of neural stem cells positive for both GFAP and Edu in the SGZ of control and Ppm1k cKO mice per 0.1 mM of coronal tissue of the dentate gyrus. Ctrl n = 5, cKO n = 5; *p < 0.05. Scale bars = 150 μm. E) Representative images from 6-month-old control and Ppm1k cKO mouse coronal hippocampal sections stained for GFAP in green, Sox2 in red, DAPI in blue. Scale bars = 150 μm. Right: Quantification of the number of cells that are GFAP and Sox2 double positive in the SGZ of control and Ppm1k cKO mice per 0.1 mM of coronal tissue of the dentate gyrus. Ctrl n = 5, cKO n = 5; **p < 0.01. All p values were determined by independent samples two-tailed Student’s t-test

Article Snippet: Rabbit DCX (Cell Signaling #4604); mouse NeuN (abcam ab104224); rabbit NeuN (Proteintech #26975-1-AP); chicken GFAP (Novus Biologicals #NBP1-05198); rabbit NeuroD1 (Proteintech #12081-1-AP); rabbit Sox2 (Genetex #GTX101507); rabbit BCKDH, (abcam #ab126173); rabbit pBCKDH (abcam #ab200577); rabbit PPM1K (Proteintech #14573-1-AP); rabbit actin (abcam #ab1801); mouse gTub, (Sigma T6557); rabbit pS6K (Cell Signaling #9234); rabbit S6K, (Cell Signaling #2708).

Techniques: Control, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Nervous system reduction in branched-chain amino acid metabolism disrupts hippocampal neurogenesis and memory

doi: 10.1101/2022.06.10.495711

Figure Lengend Snippet: A) Schematic illustrating the method used to perform scRNA sequencing. B) Illustration of the lineage stages of adult neural stem cells (NSCs) differentiation into mature neurons within the dentate gyrus; undifferentiated neural stem cells (udNSCs), activated neural stem cells (aNSCs), transit amplifying cells (TA), immature neuron (iNeu), and mature neuron (mNeu). C) Gene markers for specific neural stem cell subtypes plotted on the UMAP. Four cell type specific markers are show for each udNSCs, aNSCs, TA, and iNeu populations. D) Refined UMAP showing the four major cell types identified through marker selection. The cell population marked “other” was not identifiable by cell subtype markers. E) Comparison of refined UMAPs from control and Ppm1k KO NSC. Color is coded similar to (D). Dashed boxes outline populations increased in the knockout condition. F) PCA plots generated from refined cell subtype maps of control and PPM1K KO NSCs. Color is coded similar to (E). Arrow signifies the increase in activated NSCs and TA populations in the knockout condition. G) Pie graph showing the percentages of each cell subtype present in control and Ppm1k KO NSC cultures. H) Representative confocal images from control and Ppm1k KO NSCs labeled for GFP (green), NeuroD1 (red), and DAPI (blue). Arrows point to cells that are both positive for GFP and NeuroD1. Scale bar = 20 μm. I) Quantification of the relative mRNA expression levels for Ppm1k in control and Ppm1k KO cultures. ****p < 0.0001; Ctrl n = 6, cKO n = 6. All data are presented as mean +/- SD. J) Quantification of the percentage of iNeu cells that are positive for both GFP and NeuroD1 in control and Ppm1k KO cultures. Ctrl n = 6, cKO n = 6; *p < 0.001. All p values were determined by independent samples two-tailed Student’s t-test

Article Snippet: Rabbit DCX (Cell Signaling #4604); mouse NeuN (abcam ab104224); rabbit NeuN (Proteintech #26975-1-AP); chicken GFAP (Novus Biologicals #NBP1-05198); rabbit NeuroD1 (Proteintech #12081-1-AP); rabbit Sox2 (Genetex #GTX101507); rabbit BCKDH, (abcam #ab126173); rabbit pBCKDH (abcam #ab200577); rabbit PPM1K (Proteintech #14573-1-AP); rabbit actin (abcam #ab1801); mouse gTub, (Sigma T6557); rabbit pS6K (Cell Signaling #9234); rabbit S6K, (Cell Signaling #2708).

Techniques: Sequencing, Marker, Selection, Comparison, Control, Knock-Out, Generated, Labeling, Expressing, Two Tailed Test

Journal:

Article Title: NeuroD1/E47 Regulates the E-box Element of a Novel Zinc Finger Transcription Factor, IA-1, in Developing Nervous System *

doi: 10.1074/jbc.M306795200

Figure Lengend Snippet: A, dose-dependent activation of the IA-1 promoter by NeuroD1 in HeLa cells. Different concentrations of CMV-NeuroD1 constructs were co-transfected with the IA-1 promoter-CAT construct into HeLa cells. Dosage-dependent activation of the IA-1 promoter by the CMV-NeuroD1 cDNA expression vector is shown as -fold increase. The CAT activities were normalized by protein concentrations. The transfections were repeated three times. B, co-transfection of E47 and/or NeuroD1 enhanced the IA-1 promoter activity in HeLa cells. E47 and NeuroD1 together demonstrated much higher promoter activity than each transcription factor alone, suggesting heterodimer activation of the IA-1 promoter. The CMV-β-gal vector was used as internal control to normalize transfection efficiency. The transfections were repeated three times.

Article Snippet: Synthesis of E47 and NeuroD1 was confirmed by Western blot analysis using an anti-E47 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or an anti-NeuroD1 rabbit polyclonal antibody (CeMines).

Techniques: Activation Assay, Construct, Transfection, Expressing, Plasmid Preparation, Cotransfection, Activity Assay

Journal:

Article Title: NeuroD1/E47 Regulates the E-box Element of a Novel Zinc Finger Transcription Factor, IA-1, in Developing Nervous System *

doi: 10.1074/jbc.M306795200

Figure Lengend Snippet: Three copies of each E-box (E1, E2, and E3) were cloned into E1bTATA-CAT vector. Each reporter vector was co-transfected with pcDNA, NeuroD1, E47, or E47-NeuroD1 into HeLa cells. The E47 homodimer and the E47-NeuroD1 heterodimer showed a strong activation of the E3-box construct. The CMV-β-gal vector was used as internal control to normalize transfection efficiency. The transfections were repeated three times.

Article Snippet: Synthesis of E47 and NeuroD1 was confirmed by Western blot analysis using an anti-E47 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or an anti-NeuroD1 rabbit polyclonal antibody (CeMines).

Techniques: Clone Assay, Plasmid Preparation, Transfection, Activation Assay, Construct

Journal:

Article Title: NeuroD1/E47 Regulates the E-box Element of a Novel Zinc Finger Transcription Factor, IA-1, in Developing Nervous System *

doi: 10.1074/jbc.M306795200

Figure Lengend Snippet: A, the NeuroD1/E47 heterodimer and the E47 homodimer can specifically bind to the E3-box. The radiolabeled IA-1 promoter (−192/−165 bp) containing the E3-box was incubated with E47 or E47-NeuroD1 with or without antibodies to E47 or NeuroD1. Both the homodimer and the heterodimer were shown in shifted bands. E47 antibody supershifted the homodimer, whereas the NeuroD1 antibody interfered with the heterodimer binding. NS, nonspecific band. B, competitive inhibition of the E47-NeuroD1 complex binding with 3× E3-box. Incubation of E47 and NeuroD1 proteins with labeled 3× E3 oligonucleotide in the presence or absence of competitor (50- or 100-fold molar excess) showed that E47-NeuroD1 binding is specific for the E3-box alone.

Article Snippet: Synthesis of E47 and NeuroD1 was confirmed by Western blot analysis using an anti-E47 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or an anti-NeuroD1 rabbit polyclonal antibody (CeMines).

Techniques: Incubation, Binding Assay, Inhibition, Labeling

Journal:

Article Title: NeuroD1/E47 Regulates the E-box Element of a Novel Zinc Finger Transcription Factor, IA-1, in Developing Nervous System *

doi: 10.1074/jbc.M306795200

Figure Lengend Snippet: Twenty micrograms of total RNA from Daoy, D283MED, U87MG, WERI-Rb-1, Y79, and βTC-1 cell lines were resolved on a 1% agarose/formaldehyde gel and transferred to a nitrocellulose membrane. The samples were run as two separate sets on the same gel. The membrane was divided in half, and the left half was hybridized with a mixture of human and mouse IA-1 probes (A) and the right half was hybridized with a hamster NeuroD1/β2 probe (B). The ethidium bromide-stained gel was shown below the blot for comparison of loading differences. The IA-1 gene expression in the neuroendocrine cell lines overlaps with the NeuroD1 message completely.

Article Snippet: Synthesis of E47 and NeuroD1 was confirmed by Western blot analysis using an anti-E47 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or an anti-NeuroD1 rabbit polyclonal antibody (CeMines).

Techniques: Staining, Expressing

Journal:

Article Title: NeuroD1/E47 Regulates the E-box Element of a Novel Zinc Finger Transcription Factor, IA-1, in Developing Nervous System *

doi: 10.1074/jbc.M306795200

Figure Lengend Snippet: A, dose-dependent activation of the IA-1 promoter by NeuroD1 in HeLa cells. Different concentrations of CMV-NeuroD1 constructs were co-transfected with the IA-1 promoter-CAT construct into HeLa cells. Dosage-dependent activation of the IA-1 promoter by the CMV-NeuroD1 cDNA expression vector is shown as -fold increase. The CAT activities were normalized by protein concentrations. The transfections were repeated three times. B, co-transfection of E47 and/or NeuroD1 enhanced the IA-1 promoter activity in HeLa cells. E47 and NeuroD1 together demonstrated much higher promoter activity than each transcription factor alone, suggesting heterodimer activation of the IA-1 promoter. The CMV-β-gal vector was used as internal control to normalize transfection efficiency. The transfections were repeated three times.

Article Snippet: For supershift assay, 1 μl of a 1:5 dilution of E47 antibody (Santa Cruz Biotechnology) or a 1:2.5 dilution of NeuroD1 rabbit polyclonal antibody (CeMines) was added to the mixture following a 10-min preincubation at room temperature with in vitro translated protein and probe.

Techniques: Activation Assay, Construct, Transfection, Expressing, Plasmid Preparation, Cotransfection, Activity Assay

Journal:

Article Title: NeuroD1/E47 Regulates the E-box Element of a Novel Zinc Finger Transcription Factor, IA-1, in Developing Nervous System *

doi: 10.1074/jbc.M306795200

Figure Lengend Snippet: Three copies of each E-box (E1, E2, and E3) were cloned into E1bTATA-CAT vector. Each reporter vector was co-transfected with pcDNA, NeuroD1, E47, or E47-NeuroD1 into HeLa cells. The E47 homodimer and the E47-NeuroD1 heterodimer showed a strong activation of the E3-box construct. The CMV-β-gal vector was used as internal control to normalize transfection efficiency. The transfections were repeated three times.

Article Snippet: For supershift assay, 1 μl of a 1:5 dilution of E47 antibody (Santa Cruz Biotechnology) or a 1:2.5 dilution of NeuroD1 rabbit polyclonal antibody (CeMines) was added to the mixture following a 10-min preincubation at room temperature with in vitro translated protein and probe.

Techniques: Clone Assay, Plasmid Preparation, Transfection, Activation Assay, Construct

Journal:

Article Title: NeuroD1/E47 Regulates the E-box Element of a Novel Zinc Finger Transcription Factor, IA-1, in Developing Nervous System *

doi: 10.1074/jbc.M306795200

Figure Lengend Snippet: A, the NeuroD1/E47 heterodimer and the E47 homodimer can specifically bind to the E3-box. The radiolabeled IA-1 promoter (−192/−165 bp) containing the E3-box was incubated with E47 or E47-NeuroD1 with or without antibodies to E47 or NeuroD1. Both the homodimer and the heterodimer were shown in shifted bands. E47 antibody supershifted the homodimer, whereas the NeuroD1 antibody interfered with the heterodimer binding. NS, nonspecific band. B, competitive inhibition of the E47-NeuroD1 complex binding with 3× E3-box. Incubation of E47 and NeuroD1 proteins with labeled 3× E3 oligonucleotide in the presence or absence of competitor (50- or 100-fold molar excess) showed that E47-NeuroD1 binding is specific for the E3-box alone.

Article Snippet: For supershift assay, 1 μl of a 1:5 dilution of E47 antibody (Santa Cruz Biotechnology) or a 1:2.5 dilution of NeuroD1 rabbit polyclonal antibody (CeMines) was added to the mixture following a 10-min preincubation at room temperature with in vitro translated protein and probe.

Techniques: Incubation, Binding Assay, Inhibition, Labeling

Journal:

Article Title: NeuroD1/E47 Regulates the E-box Element of a Novel Zinc Finger Transcription Factor, IA-1, in Developing Nervous System *

doi: 10.1074/jbc.M306795200

Figure Lengend Snippet: Twenty micrograms of total RNA from Daoy, D283MED, U87MG, WERI-Rb-1, Y79, and βTC-1 cell lines were resolved on a 1% agarose/formaldehyde gel and transferred to a nitrocellulose membrane. The samples were run as two separate sets on the same gel. The membrane was divided in half, and the left half was hybridized with a mixture of human and mouse IA-1 probes (A) and the right half was hybridized with a hamster NeuroD1/β2 probe (B). The ethidium bromide-stained gel was shown below the blot for comparison of loading differences. The IA-1 gene expression in the neuroendocrine cell lines overlaps with the NeuroD1 message completely.

Article Snippet: For supershift assay, 1 μl of a 1:5 dilution of E47 antibody (Santa Cruz Biotechnology) or a 1:2.5 dilution of NeuroD1 rabbit polyclonal antibody (CeMines) was added to the mixture following a 10-min preincubation at room temperature with in vitro translated protein and probe.

Techniques: Staining, Expressing