Journal: PNAS nexus

Article Title: Genetic modulation of the HTR2A gene reduces anxiety-related behavior in mice.

doi: 10.1093/pnasnexus/pgad170

Figure Lengend Snippet: Fig. 2. In vitro exposure of AAV copackaged Cas9 DNA with HTR2A-targeting gRNA leads to a decrease in spontaneous electrical activity of primary mouse cortical neurons. MEA analysis was performed in mouse cortical neurons following treatment with a mixture of AAV9 vectors containing AAV9– Mecp2–spCas9–sPA and AAV9–GFP–U6–mHtr2a–gRNA–ssODN at various concentrations indicated by the MOI. Neurons were treated at day 6, and MEA analyses were performed on day 14 (see the Methods section for details). A) Transduction was confirmed in primary cortical neurons by detecting GFP fluorescence. As shown, strong GFP fluorescence was observed at a MOI of 2 × 105, with the intensity decreasing significantly at 2 × 104, and 2 × 103 MOI, respectively. B) There are 16 electrodes in each well, and each panel represents the recording of the real-time signal of one selected electrode at different MOIs. The gray threshold lines represent the voltage of the baseline noise. If an electrode signal is beyond the threshold, it will be recorded as a spike. As the MOI increased, the number of spikes decreased significantly as compared with vehicle. C) Shown are continuous spike signals of all 16 electrodes within 50 s. Each row presents an electrode. The bursts are shown as vertical bars. A network of bursts is a coordinated cluster of spiking across multiple electrodes. There were network bursts in the vehicle and lowest MOI, but not at higher MOIs including 2 × 104 and 2 × 105. D–F) Quantification of MEA analysis showing the total number of spikes over the duration of the analysis D), the number of network bursts defined as a cluster of spikes across all electrodes E), and the synchrony index, which indicates a unitless measure of synchrony between 0 and 1. Values closer to 1 indicate higher synchrony. In all cases, exposure of neurons to 2 × 105 MOI led to a significant decrease in the number of spikes (83% decrease compared with vehicle controls, P = 0.0007, N = 3) D); in the number of bursts (68% decrease compared with vehicle controls, P = 0.014, N = 3) E); and a decrease in the synchrony index (34% decrease compared with vehicle controls, P = 0.0002, N = 3) F). G) and H) are identical in terms of experimental procedure as A–F), with the exception that genomic DNA was collected following treatments and next-generation targeted sequencing was performed as described in the Methods section. G) depicts the efficiency analysis of gene editing in a stacked column graph with the total number of reads (unmapped versus mapped) for each treatment group. The unmapped reads represent the counts of sequence that were inconsistent with the reference sequence (HTR2A gene). The mapped reads represent the counts of sequence that were consistent with the reference sequence. At the highest concentration, MOI = 2 × 105, a 41.4% gene editing efficiency was observed, followed by a 2% at MOI of 2 × 104. In H), indels within the target sequence of the HTR2A gene (149 bp) are shown with the top panel representing the insertion ratio, while the bottom pattern representing the deletion ratio. In both cases, the highest MOI concentration led to a sharp increase in indel formation within the target site. NGG in both graphs indicates the PAM sequence. AAV9 volumes of 40, 4, and 0.4 µL correspond to a MOI of 2 × 105, 2 × 104, and 2 × 103, respectively (see Table 1 of main text).

Article Snippet: The plasmid design for expression of spCas9 under the neuronal-specific promoter, MeCP2, was from Addgene (17).

Techniques: In Vitro, Activity Assay, Transduction, Fluorescence, Sequencing, Concentration Assay

Journal: PNAS nexus

Article Title: Genetic modulation of the HTR2A gene reduces anxiety-related behavior in mice.

doi: 10.1093/pnasnexus/pgad170

Figure Lengend Snippet: Fig. 3. Intranasal delivery of AAV9 vectors containing Cas9 and gRNA receptor leads to down-regulation of 5HT-2A receptor mRNA. A) Experimental workflow (see Methods for details). AAV9–Mecp2–spCas9–sPA and AAV9–GFP–U6–mHtr2a–gRNA–ssODN were synthesized and typical viral titers were on the order of 2.0 × 1013 GC/mL. The plasmid design for expression of spCas9 under the neuronal-specific promoter, MeCP2, was from Addgene (17). Separately, AAV9 vectors were constructed that contained the gRNA for the HTR2A gene with a GFP coexpression system. Both vectors were mixed at equal concentrations and delivered intranasally via a micropipette tip (∼2.0 × 1011 viral particles). B) Representative, low-field immunofluorescence sagittal image following treatment, fixation in formalin, and immunolabeling using a specific antibody against GFP (1:1,000). GFP-positive neurons positively transfected with gRNA were identified in most brain regions including the olfactory bulb (OB), cortex, cerebellum, and numerous subcortical areas including the IPN, a major connectome for stress-mediated pathways. C) Data show the results of qPCR real-time assays to analyze mRNA levels of Htr2a following extraction of total brain RNA from frozen brain tissue in either vehicle controls (green bar) or CRISPR/Cas9 treated (bar labeled ‘treated’). Results display the relative change in expression. Real-time PCR results represent a total of N = 5 animals for each group performed in triplicate ± SEM. Asterisk denotes significant difference between the two groups, P ≤0.05. D) NGS analyses on-target effects in five individual mouse brain samples were analyzed and the most prevalent reads identified are shown. The profile mutations induced by HTR2A-targeting AAV-CRISPR/Cas9 revealed single base pair deletions indicated by the −1 symbol in adenine, which occurred in the exact positions for all five animals. E) The corresponding predicted amino acid sequences are presented for the two adenine indels with both mutations leading to a nonsense introduction of a premature stop codon. *Denotes single base pair deletion of adenine closest to the PAM sequnce. **Denotes single base pair deletion of adenine farthest from the PAM sequence. PAM, protospacer adjacent motif; WT, wild type.

Article Snippet: The plasmid design for expression of spCas9 under the neuronal-specific promoter, MeCP2, was from Addgene (17).

Techniques: Synthesized, Plasmid Preparation, Expressing, Construct, Immunofluorescence, Immunolabeling, Transfection, Extraction, CRISPR, Labeling, Real-time Polymerase Chain Reaction, Sequencing

Journal: Diabetes

Article Title: Fat-Specific Knockout of Mecp2 Upregulates Slpi to Reduce Obesity by Enhancing Browning.

doi: 10.2337/db19-0502

Figure Lengend Snippet: Fig. 1 Mecp2 is upregulated in WATs of obese humans/mice and during in vivo adipogenesis. (A-C) qPCR analysis (A), representative images (B) and western blots (left) with quantitative results (right) (C) of Mecp2 in eWAT of NC- or six-month HFD-fed male mice. (n = 5-6 per mouse group). (D) qPCR analysis of MECP2 in sWAT of human subjects (normal human subjects (n = 17)) and obese human subjects (n = 15). (E) Correlation between MECP2 and body mass index (BMI). (F-G) western blots (F; normal human subjects, n = 5) and obese human subjects (n = 7) and representative images (G; normal human subjects, n = 10) and obese human subjects (n = 12) of MECP2 in sWAT of human subjects. (H-K) Increased Mecp2 levels during white adipocyte differentiation in SVF and 3T3-L1 cells. The representative mRNA levels of indicated genes (H and J) and representative western blots (left) with quantitative results (right) of Pparγ1/2, Fabp4 and Mecp2 (I and K) during SVF and 3T3-L1 white differentiation, respectively. * P < 0.05; * * P < 0.01.

Article Snippet: To overexpress Mecp2 in 3T3-L1 cells, cells was transfected with plasmid pCMV6-mMecp2-Flag (Origene).

Techniques: In Vivo, Western Blot

Journal: Diabetes

Article Title: Fat-Specific Knockout of Mecp2 Upregulates Slpi to Reduce Obesity by Enhancing Browning.

doi: 10.2337/db19-0502

Figure Lengend Snippet: Fig. 6 Mecp2 negatively regulates Slpi level. (A) Volcanic map of several highly upregulated genes in iWAT of Mecp2Adi KO mice. (B and C) Slpi level in iWAT (B) and SVF or mature adipocytes of iWAT (C) of WT or

Article Snippet: To overexpress Mecp2 in 3T3-L1 cells, cells was transfected with plasmid pCMV6-mMecp2-Flag (Origene).

Techniques:

Journal: Diabetes

Article Title: Fat-Specific Knockout of Mecp2 Upregulates Slpi to Reduce Obesity by Enhancing Browning.

doi: 10.2337/db19-0502

Figure Lengend Snippet: Fig. 7 Mecp2 regulates browning by Slpi. (A) Experimental design and iWAT AAVs injection. (B) mRNA level of Slpi in iWAT injected with AAV-shSlpi or AAV-Scram. (C) Weights of iWAT. (D-E) Representative images of H&E/Ucp1 staining (D) and representative western blots (left) with quantitative results (right) of Ucp1 (E) in iWAT of WT or Mecp2Adi KO mice after 4◦C stress. (F) mRNA levels of thermogenic genes in iWAT of WT or Mecp2Adi KO mice after 4◦C stress. (G) serum Slpi level after iWAT pads of both flanks injected with AAV- shSlpi or AAV-Scram. n = 3-7 per group , * P < 0.05; * * P < 0.01.

Article Snippet: To overexpress Mecp2 in 3T3-L1 cells, cells was transfected with plasmid pCMV6-mMecp2-Flag (Origene).

Techniques: Injection, Staining, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: Whole Genome Expression Analysis in a Mouse Model of Tauopathy Identifies MECP2 as a Possible Regulator of Tau Pathology

doi: 10.3389/fnmol.2017.00069

Figure Lengend Snippet: Microarray analysis of the hippocampi of 6-month-old WT and hTau MaptKO ( Duke ) mice. (A) Total RNA from hippocampi of 6-month-old WT and hTau MaptKO ( Duke ) mice was hybridized to MouseWG-6 v2.0 Expression BeadChip (Illumina ® ). Expression of 64 genes that were significantly altered in hTau MaptKO ( Duke ) mice compared to WT is shown. Values displayed as fold change in expression level: up-regulated (red) and down-regulated (blue) genes. Data represents mean fold change from three mice per genotype. (B) Gene interaction network analysis (using Metacore analytical suite) for the 64 significantly altered genes in hTau MaptKO ( Duke ) mice compared to WT mice. The genes with red (or blue) next to their graphic key is either up- (or down-) regulated. As expected, endogenous mouse tau (MAPT) is one of the most down-regulated genes. Other genes relevant to neuronal function or neurological disease include Prkca, Mecp2, Strn4, Slc40a1, Pold2, Pcsk2 (up-regulated) and Krt12, Lass1, Plat and Nrxn1 (down-regulated). Many of the altered genes are regulated by the transcription factor SP1. (C) Venn diagrams showing GO’s biological processes when all 64 genes are categorized for top biological processes segregate into three distinct Venn diagrams: Group 1: anatomical structure development – 17 altered genes out of total 47, i.e., 17/47; cellular nitrogen compound metabolic process – 15/47; biosynthetic process 13/47. Group 2: cell death – 9/47; signal transduction 8/47; response to stress 8/47. Group 3: neurological system 5/47, cell cycle 2/47 and immune system processes 4/47. Note the genes written in red (or blue) are significantly up- (or down-) regulated.

Article Snippet: Other antibodies: GAPDH (rabbit polyclonal antibody; EMD Millipore; #ABS16), T-MECP2 (D4F3; rabbit monoclonal antibody; Cell Signaling #3456); phospho-MECP2 (pSer80; rabbit polyclonal antibody; PhosphoSolutions #p1205-80).

Techniques: Microarray, Expressing, Transduction

Journal: Frontiers in Molecular Neuroscience

Article Title: Whole Genome Expression Analysis in a Mouse Model of Tauopathy Identifies MECP2 as a Possible Regulator of Tau Pathology

doi: 10.3389/fnmol.2017.00069

Figure Lengend Snippet: MECP2 expression and phosphorylation is up-regulated in 6-month-old hTau MaptKO ( Duke ) mice. (A) qRT-PCR analysis showing statistically significant ( ∗ p < 0.05; unpaired t- test; n = 4 WT, and n = 4 for hTau MaptKO ( Duke ) mice; mean + SEM) up-regulation of MECP2 in the hemi-brains of 6-month-old hTau MaptKO ( Duke ) vs. WT mice. (B) Note the regional differences in the expression of MECP2 in the hippocampus (HIP), cortex (CX), and rest of the brain (ROB) that are devoid of CX and HP. (C) Another gene ( Vat1l ) that was increased in our whole genome microarray analysis also showed modest up-regulation in its mRNA levels in the brains of hTau MaptKO ( Duke ) mice compared to age-matched WT mice. (D,E) Western blot analysis showing a trend toward increased levels for phospho(p)-Ser80 MECP2/total (t) MECP2 and tMECP2/GAPDH [ p = 0.08; unpaired t- test; n = 3, all females for WT and n = 10, three females and six males for hTau MaptKO ( Duke ) ] in 6-month-old hTau MaptKO ( Duke ) versus WT mice; mean + SEM). (F) Double IF and confocal microscopy analysis revealing a modest increase in pMECP2 in the CA3 region of HP in 6-month-old hTau MaptKO ( Duke ) mice compared to age-matched WT controls. Scale bar 25 μm.

Article Snippet: Other antibodies: GAPDH (rabbit polyclonal antibody; EMD Millipore; #ABS16), T-MECP2 (D4F3; rabbit monoclonal antibody; Cell Signaling #3456); phospho-MECP2 (pSer80; rabbit polyclonal antibody; PhosphoSolutions #p1205-80).

Techniques: Expressing, Phospho-proteomics, Quantitative RT-PCR, Microarray, Western Blot, Confocal Microscopy

Journal: Frontiers in Molecular Neuroscience

Article Title: Whole Genome Expression Analysis in a Mouse Model of Tauopathy Identifies MECP2 as a Possible Regulator of Tau Pathology

doi: 10.3389/fnmol.2017.00069

Figure Lengend Snippet: MECP2 phosphorylation is up-regulated in 12-month-old hTau MaptKO ( Duke ) mice and in human AD brain. (A,B) Western blot analysis showing pMECP2/GAPDH ratio significantly higher [ ∗ p < 0.05; unpaired t- test; n = 3 for hTau MaptKO ( Duke ) versus WT mice; all three males for WT; two males and one female for hTau MaptKO ( Duke ) ; mean + SEM] in the hippocampus of 12-month-old hTau MaptKO ( Duke ) mice compared to WT controls. No alteration in the tMECP2/GAPDH ratio in the hippocampus of 12-month-old hTau MaptKO ( Duke ) mice compared to controls. (C) Significantly elevated pMECP2 immunoreactive specks within the nucleus of CA3 hippocampal neurons of 12-month-old hTau MaptKO ( Duke ) mice compared to age-matched WT controls. (D) Double IF and confocal microscopy analysis shows a modest increase in the pMECP2 in the CA3 neuronal layer of 12-month-old hTau MaptKO ( Duke ) mice compared to age-matched WT controls. (E–G) Confocal projections (in E,G ) and bright field images show elevated pMECP2 immunoreactivity and co-localization with nuclear stain DAPI (in E,G ) or peri-vascular labeling (in F ) specifically in the Layer III of temporal cortex of human AD brain autopsy sections compared to the to non-AD healthy control subject. Orthogonal view in (G) shows pMECP2 labeling to be nuclear or peri-nuclear in the human AD cortex. Scale bar 10 μm (in C,F ); 25 μm (in D,G ); 100 μm (in E ).

Article Snippet: Other antibodies: GAPDH (rabbit polyclonal antibody; EMD Millipore; #ABS16), T-MECP2 (D4F3; rabbit monoclonal antibody; Cell Signaling #3456); phospho-MECP2 (pSer80; rabbit polyclonal antibody; PhosphoSolutions #p1205-80).

Techniques: Phospho-proteomics, Western Blot, Confocal Microscopy, Staining, Labeling, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: Whole Genome Expression Analysis in a Mouse Model of Tauopathy Identifies MECP2 as a Possible Regulator of Tau Pathology

doi: 10.3389/fnmol.2017.00069

Figure Lengend Snippet: MECP2 regulates tau pathology in vitro . N2a cells transiently transfected with human tau 0N3R isoform (‘+Tau’) or a control plasmid (‘-Tau’) were nucleofected with siRNA [scramble siRNA (siScr) or MECP2 siRNA]. After 24 h of siRNA nucleofection, the cells were harvested and probed for AT180, PHF-1, Tau5, Tau12, and T-MECP2. (A,B) Note that siMECP2 significantly reduced levels of MECP2 in both ‘-Tau’ and ‘+Tau’ N2a cells ( ∗ p < 0.01; unpaired t- test; n = 3 replicates; mean + SEM). (C,D) siMECP2 treatment also significantly ( ∗ p < 0.01; unpaired t- test; n = 3 replicates; mean + SEM) reduced the levels of both total tau (Tau5/GAPDH) and human tau (Tau12/GAPDH) ratios in the ‘+Tau’ N2a cells compared to scramble siRNA treated conditions. (E–H) siMECP2 knockdown resulted in statistically significant ( ∗ p < 0.01; unpaired t- test; n = 3 replicates; mean + SEM) increase and decrease in AT180/Tau5 and PHF1/Tau5 ratios, respectively. Note that the ratio for β-actin/GAPDH was not altered either in ‘-Tau’/‘+Tau’ conditions or with/without siMECP2 conditions.

Article Snippet: Other antibodies: GAPDH (rabbit polyclonal antibody; EMD Millipore; #ABS16), T-MECP2 (D4F3; rabbit monoclonal antibody; Cell Signaling #3456); phospho-MECP2 (pSer80; rabbit polyclonal antibody; PhosphoSolutions #p1205-80).

Techniques: In Vitro, Transfection, Control, Plasmid Preparation, Knockdown

Journal: Cancer cell

Article Title: The osteogenic niche is a calcium reservoir of bone micrometastases and confers unexpected therapeutic vulnerability

doi: 10.1016/j.ccell.2018.10.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Western blots were performed using antibodies against NFAT1(Cell Signalling, 5861S), pS6K(T389) (Cell Signaling, 9234S), pCamKII(T286) (Cell Signalling, 12716S), MeCP2 (Cell Signalling, 3456S), pMeCP2(S421)(Rockland, 600-401-X14), Firefly luciferase (Thermo, PA5-32209).

Techniques: Luciferase, Recombinant, Expressing, In Vivo, Software