Journal: Neuron

Article Title: Genome-wide in vivo CNS Screening Identifies Genes that Modify CNS Neuronal Survival and mHTT Toxicity

doi: 10.1016/j.neuron.2020.01.004

Figure Lengend Snippet: Key Resources Table:

Article Snippet: Human samples: Human TissueScan cDNA arrays (Origene, Rockville, MD) composed of 24 different brain regions from normal, de-identified post mortem human brain tissue were used for quantification of relative gene expression across brain regions as described in the qRT-PCR Method Detail.

Techniques: Virus, Genome Wide, shRNA, Control, Sequencing, Recombinant, Expressing, Multiplex Assay, Software

Journal: Scientific Reports

Article Title: Different risk and protective factors predict change of planning ability in middle versus older age

doi: 10.1038/s41598-024-76784-1

Figure Lengend Snippet: Overview of the assessed parameters and used instruments.

Article Snippet: Genotyping and imputation of Single Nucleotide Polymorphisms , KIBRA rs17070145 BDNF rs6265 DBH rs1611115 DRD1 rs4532 ANKK1 rs1800497 DRD2 rs6277 COMT rs4680 COMT rs4818 APOE rs429358 APOE rs7412 , Affymetrix Genome-Wide Human SNP 6.0 array (Affymetrix, Santa Clara, CA) . APOE SNPs (rs429358 and rs7412) were aggregated accordingly to include the epsilon 2, 3 and 4 variants in the analyses. .

Techniques: Infection, Biomarker Discovery, Genome Wide

Journal: Scientific Reports

Article Title: Different risk and protective factors predict change of planning ability in middle versus older age

doi: 10.1038/s41598-024-76784-1

Figure Lengend Snippet: Overview of the SNPs of interest with the respective chromosomes, genes and the corresponding allele variants and the absolute number of observations.

Article Snippet: Genotyping and imputation of Single Nucleotide Polymorphisms , KIBRA rs17070145 BDNF rs6265 DBH rs1611115 DRD1 rs4532 ANKK1 rs1800497 DRD2 rs6277 COMT rs4680 COMT rs4818 APOE rs429358 APOE rs7412 , Affymetrix Genome-Wide Human SNP 6.0 array (Affymetrix, Santa Clara, CA) . APOE SNPs (rs429358 and rs7412) were aggregated accordingly to include the epsilon 2, 3 and 4 variants in the analyses. .

Techniques:

Journal: bioRxiv

Article Title: Loss of Dnmt3a dependent methylation in inhibitory neurons impairs neural function through a mechanism that impacts Rett syndrome

doi: 10.1101/815639

Figure Lengend Snippet: A) Western blot of half brain hemisphere from Dnmt3a or Mecp2 cKO mice demonstrating appropriate loss of each protein. B) Immunofluorescence (IF) images of WT, Dnmt3a cKO or Mecp2 cKO mice probing for Dnmt3a or MeCP2 (red), the Sun1-sfGFP-myc-PA fusion protein that marks the nuclear envelope and is dependent on Cre expression (green), and DAPI to mark genomic DNA (blue). n=3 mice per genotype (western), n=3 mice (IF) representative image shown.

Article Snippet: All sections were blocked in blocking buffer (2% normal goat serum, 0.3% Triton X-100, in 1XPBS) for 1hr at RT and then stained with respective primary antibodies in blocking buffer (1:250 anti-Dnmt3a (VWR 64B1446) and 1:200 anti-Myc (Sigma C3956) for 2-week old brains and 1:500 anti-MeCP2 (Cell signaling 3456) and 1:800 anti-GFP (abcam 13970) for 6-week old brains).

Techniques: Western Blot, Immunofluorescence, Expressing

Journal: bioRxiv

Article Title: Loss of Dnmt3a dependent methylation in inhibitory neurons impairs neural function through a mechanism that impacts Rett syndrome

doi: 10.1101/815639

Figure Lengend Snippet: A) Mice that lack Dnmt3a or MeCP2 in inhibitory neurons present with hindlimb spasticity. B) Obsessive grooming is increased in both knockout models. C) Nest building, D) grip strength, E) Open Field, F) Fear conditioning tests revealed impairments in both cKO lines. G ) Self-injury in Dnmt3a cKO and Mecp2 cKO mice necessitated humane euthanasia. H) Example traces of miniature inhibitory postsynaptic currents (mIPSCs) recorded in the dorsal striatum. Both cKO models show similar alterations in I) amplitude. J) Weekly body weight records for Dnmt3a cKO and Mecp2 cKO mice showed only Dnmt3a cKO mice (here separated by sex-see Methods ) were runted. K ) Dnmt3a cKO and Mecp2 cKO mice showed opposite alterations in acoustic startle response. L) Only Mecp2 cKO mice displayed impairment on the parallel rod. M) Dnmt3a cKO mice had to undergo earlier euthanasia than Mecp2 cKO mice due to the severity of their self-lesioning. n= 11-52 (behavior), n=5-9mice per genotype with 24-50 neurons total (electrophysiology). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See Supplemental Table 1 for full statistics .

Article Snippet: All sections were blocked in blocking buffer (2% normal goat serum, 0.3% Triton X-100, in 1XPBS) for 1hr at RT and then stained with respective primary antibodies in blocking buffer (1:250 anti-Dnmt3a (VWR 64B1446) and 1:200 anti-Myc (Sigma C3956) for 2-week old brains and 1:500 anti-MeCP2 (Cell signaling 3456) and 1:800 anti-GFP (abcam 13970) for 6-week old brains).

Techniques: Knock-Out

Journal: bioRxiv

Article Title: Loss of Dnmt3a dependent methylation in inhibitory neurons impairs neural function through a mechanism that impacts Rett syndrome

doi: 10.1101/815639

Figure Lengend Snippet: A ) Both mouse lines showed decreased rearing in the Open Field test. B) Dnmt3a cKO mice showed impaired fear learning, whereas Mecp2 cKO mice did not differ from control mice. C) Cue memory was normal in both cKO mice. D-F) Tests for anxiety-like behaviors (open field, light dark or elevated plus maze, respectively). G) Hot plate and H) tail flick testing for nociceptive pain in both cKO mice. I) Rotarod test for motor learning and coordination and J) the partition test for social interaction. K-N ) Frequency, rise and decay measures from mIPSCs from the striatum. O) Only Mecp2 cKO had a trend for increased pre-pulse inhibition. n= 11-50 per genotype (behavior), n= 5-9 mice per genotype with 24-50 neurons total (electrophysiology), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See Supplemental Table 1 for full numbers and statistics.

Article Snippet: All sections were blocked in blocking buffer (2% normal goat serum, 0.3% Triton X-100, in 1XPBS) for 1hr at RT and then stained with respective primary antibodies in blocking buffer (1:250 anti-Dnmt3a (VWR 64B1446) and 1:200 anti-Myc (Sigma C3956) for 2-week old brains and 1:500 anti-MeCP2 (Cell signaling 3456) and 1:800 anti-GFP (abcam 13970) for 6-week old brains).

Techniques: Tail Flick Test, Inhibition

Journal: bioRxiv

Article Title: Loss of Dnmt3a dependent methylation in inhibitory neurons impairs neural function through a mechanism that impacts Rett syndrome

doi: 10.1101/815639

Figure Lengend Snippet: A) Schematic of INTACT method used to isolate inhibitory neurons from the striata of WT, Dnmt3a cKO, or Mecp2 cKO mice that also conditionally express the INTACT allele. B) Spearman correlation of methylation profiles from inhibitory neurons sorted from WT vs. Mecp2 cKO striatum, showing that methylation is stable in the absence of MeCP2. C) Bar graph showing the global mCH level in each biological replicate (left). Example genes from DNA methylome sequencing tracks showing mCH signal in two biological replicates per genotype (right). D) Bar graph showing the global mCG level in each biological replicate (left). Example genes from DNA methylome sequencing tracks showing mCG signal (right). Red bars indicate DMRs. E) Genome wide correlation between mCH and mCG in wild-type mice showing poor correlation. F) Genome wide correlation of Dnmt3a dependent mCG and mCG (the change in mCH methylation observed in the Dnmt3a cKO is defined as “Dnmt3a dependent”) showing good correlation to indicate that mCH and mCG written by Dnmt3a are coupled. Correlation values for E and F are Pearson correlations designated as “rho”. n= 2 mice per genotype (see methods for specific genotype information). See Supplemental Table 1 for replicate statistics.

Article Snippet: All sections were blocked in blocking buffer (2% normal goat serum, 0.3% Triton X-100, in 1XPBS) for 1hr at RT and then stained with respective primary antibodies in blocking buffer (1:250 anti-Dnmt3a (VWR 64B1446) and 1:200 anti-Myc (Sigma C3956) for 2-week old brains and 1:500 anti-MeCP2 (Cell signaling 3456) and 1:800 anti-GFP (abcam 13970) for 6-week old brains).

Techniques: Methylation, Sequencing, Genome Wide

Journal: bioRxiv

Article Title: Loss of Dnmt3a dependent methylation in inhibitory neurons impairs neural function through a mechanism that impacts Rett syndrome

doi: 10.1101/815639

Figure Lengend Snippet: Plot of the difference in genome wide mCH versus mCG methylation between the WT and MeCP2 cKO mice. Person correlation designated as rho. Consistent with stable methylation in the absence of MeCP2, the differences center at 0.

Article Snippet: All sections were blocked in blocking buffer (2% normal goat serum, 0.3% Triton X-100, in 1XPBS) for 1hr at RT and then stained with respective primary antibodies in blocking buffer (1:250 anti-Dnmt3a (VWR 64B1446) and 1:200 anti-Myc (Sigma C3956) for 2-week old brains and 1:500 anti-MeCP2 (Cell signaling 3456) and 1:800 anti-GFP (abcam 13970) for 6-week old brains).

Techniques: Genome Wide, Methylation

Journal: bioRxiv

Article Title: Loss of Dnmt3a dependent methylation in inhibitory neurons impairs neural function through a mechanism that impacts Rett syndrome

doi: 10.1101/815639

Figure Lengend Snippet: A) RNA-seq data of sorted WT vs Dnmt3a cKO or Mecp2 cKO striatal inhibitory neurons that also express the INTACT allele. Red dots represent genes with altered expression in the knockout cells (padj < 0.01). B) Differentially expressed genes (DEGs) that overlap between knockout models. Inhibitory neurons that lack MeCP2 or Dnmt3a share about 40% of the same DEGs. Only ∼12% of DEGs in inhibitory neurons that lack Dnmt3a are shared with neurons that lack MeCP2. C) Plot of log2 fold-change for DEGs in Dnmt3a cKO and Mecp2 cKO models. DEGs that are only significantly misregulated in the Dnmt3a cKO model, only significantly misregulated in the Mecp2 cKO model, or common to both models are colored in blue, green, or orange, respectively. The plot shows that the DEGs common to both models have similar degree and direction of change.

Article Snippet: All sections were blocked in blocking buffer (2% normal goat serum, 0.3% Triton X-100, in 1XPBS) for 1hr at RT and then stained with respective primary antibodies in blocking buffer (1:250 anti-Dnmt3a (VWR 64B1446) and 1:200 anti-Myc (Sigma C3956) for 2-week old brains and 1:500 anti-MeCP2 (Cell signaling 3456) and 1:800 anti-GFP (abcam 13970) for 6-week old brains).

Techniques: RNA Sequencing Assay, Expressing, Knock-Out

Journal: bioRxiv

Article Title: Loss of Dnmt3a dependent methylation in inhibitory neurons impairs neural function through a mechanism that impacts Rett syndrome

doi: 10.1101/815639

Figure Lengend Snippet: A) Gene-body mCH levels in different categories of DEGs in WT mice are plotted, demonstrating that misregulated genes have higher mCH than genes that are unchanged. B) Gene-body mCG levels in different categories of DEGs in WT mice are plotted, demonstrating that misregulated genes (with the exception of MeCP2 down-regulated genes) have higher mCG than genes that are unchanged. C) Running average plot of log2fold change in gene expression for genes significantly misregulated only in the Dnmt3a cKO model versus the change in mCH methylation observed in the Dnmt3a cKO model (“Dnmt3a dependent mCH methylation”) (left). Running average plot of log2fold change in gene expression for these same genes versus the change in mCG methylation observed in the Dnmt3a cKO (“Dnmt3a dependent mCG methylation”) (right). D) Running average plots of log2fold change in gene expression in the Dnmt3a cKO model for genes commonly misregulated in both cKO models versus Dnmt3a dependent mCH (right) and mCG (left). E) Running average plots of log2fold change in gene expression in the MeCP2 cKO for genes commonly misregulated in both cKO models versus Dnmt3a dependent mCH (right) and mCG (left). F) Running average plots of log2fold change in gene expression for genes that are only significantly misregulated in the MeCP2 cKO model. G-J) R 2 values from analysis in panels C-F shown as blue, orange or green dots, respectively, plotted over 1,000 random repetitions of the analysis with each repetition containing the same number of non-DEGs (padj>0.01). The results of random repetitions are shown as grey dots. All plots were made with DEGs padj<0.01. n= 2 mice per genotype for methylation data. n= 4 mice per genotype (RNA-seq). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 .

Article Snippet: All sections were blocked in blocking buffer (2% normal goat serum, 0.3% Triton X-100, in 1XPBS) for 1hr at RT and then stained with respective primary antibodies in blocking buffer (1:250 anti-Dnmt3a (VWR 64B1446) and 1:200 anti-Myc (Sigma C3956) for 2-week old brains and 1:500 anti-MeCP2 (Cell signaling 3456) and 1:800 anti-GFP (abcam 13970) for 6-week old brains).

Techniques: Expressing, Methylation, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Loss of Dnmt3a dependent methylation in inhibitory neurons impairs neural function through a mechanism that impacts Rett syndrome

doi: 10.1101/815639

Figure Lengend Snippet: A) Gene-body mCH levels in different categories of DEGs in WT, Dnmt3a cKO and Mecp2 cKO mice. B) Gene-body mCG levels in different categories of DEGs in WT, Dnmt3a cKO and Mecp2 cKO mice. C) P-value matrix for comparison of mCH levels of different categories of genes illustrating significant differences versus non-DEGs. The boxes are colored such that more significant p-values are darker shades of red. D) P-value matrix for comparison of mCG levels of different categories of genes illustrating significant differences versus non-DEGs. As in C, the boxes are colored such that more significant p-values are darker shades of red.

Article Snippet: All sections were blocked in blocking buffer (2% normal goat serum, 0.3% Triton X-100, in 1XPBS) for 1hr at RT and then stained with respective primary antibodies in blocking buffer (1:250 anti-Dnmt3a (VWR 64B1446) and 1:200 anti-Myc (Sigma C3956) for 2-week old brains and 1:500 anti-MeCP2 (Cell signaling 3456) and 1:800 anti-GFP (abcam 13970) for 6-week old brains).

Techniques:

Journal: bioRxiv

Article Title: Loss of Dnmt3a dependent methylation in inhibitory neurons impairs neural function through a mechanism that impacts Rett syndrome

doi: 10.1101/815639

Figure Lengend Snippet: The percentage of DEGs that overlap between parvalbumin (PV) and vasoactive intestinal polypeptide (VIP) neurons in the mouse cortex from Dnmt3a cKO (Nestin-Cre) and Mecp2 KO mouse models. The data are a re-analysis of single-nuclear RNA sequencing . The percentages are shown as all genes and genes broken down by direction of change (down- or up-regulated) for A) PV and VIP neurons. Consistent with our data there are few DEGs significantly misregulated in Dnmt3a cKO mice that are also significantly misregulated in the same neurons that lack MeCP2. Notably, our data show a significantly higher overlap of genes (40%) misregualted in Mecp2 cKO that are also misregulated in the Dnmt3a cKO.

Article Snippet: All sections were blocked in blocking buffer (2% normal goat serum, 0.3% Triton X-100, in 1XPBS) for 1hr at RT and then stained with respective primary antibodies in blocking buffer (1:250 anti-Dnmt3a (VWR 64B1446) and 1:200 anti-Myc (Sigma C3956) for 2-week old brains and 1:500 anti-MeCP2 (Cell signaling 3456) and 1:800 anti-GFP (abcam 13970) for 6-week old brains).

Techniques: RNA Sequencing Assay