usp4 mouse  (Proteintech)

Journal: eneuro

Article Title: Amyloid Precursor Protein (APP) Controls the Expression of the Transcriptional Activator Neuronal PAS Domain Protein 4 (NPAS4) and Synaptic GABA Release

doi: 10.1523/eneuro.0322-19.2020

Figure Lengend Snippet: Figure 1. APP-dependent expression of NPAS4 in young differentiating neuronal culture. Summary of transcriptome analysis per- formed with the GeneChip Mouse Transcriptome Array 1.0 (Affymetrix). The characterization of the model and the experimental workflow are described in Extended Data Figure 1-1. Data were processed in triplicate (N = 3) for each experimental time point (E18, DIV3, DIV7). Non-coding transcripts and alternative splicing products were detected by these arrays, but only transcripts of coding transcripts have been considered here. For all the transcripts, adjusted p . 0.05 except for APP (internal control, p , 0.05). A, Number of upregulated and downregulated coding transcript in APP/ versus APP1/1 primary neurons at E18, DIV3, and DIV7. Linear fold changes have been set at 1.25, 1.5, and 2. B, KEGG pathway analysis (http://www.genome.jp/kegg/pathway.html) at E18, DIV3, DIV7 (APP/ vs APP1/1) to identify networks molecular pathways (or interaction networks) in which differentially ex- pressed genes are clustered. The five most modified pathways are displayed for each time point, with the number of genes poten- tially lated or downregulated. C, IEGs expression in APP/ versus APP1/1 primary neurons at DIV7 and their respective fold change (APP/ vs APP1/1) in microarray analysis at DIV7. D, Neuronal PAS 4 domain (NPAS4) mRNA level was measured by qPCR at E18, DIV3, and DIV7 (n = 6, N = 3). Results (mean 6 SEM) are expressed as percentage of controls (APP1/1); n.s. = non-significant, pp = 0.0242, Student’s t test. mRNA levels of two other IEGs (Egr1 and Egr3) were meas- ured in the same conditions (Extended Data Figure 1-2).

Article Snippet: A lentiviral vector-based approach was also used to deliver the CRISPR-Cas9 system. sgRNAs Oligo2 and Oligo17 were designed using on/off-target score algorithm to target the APP mouse gene (gene ID: 11820), and sgRNA CRISPR-NPAS4 to target the NPAS4 mouse gene (gene ID: 225872). sgRNAs were cloned in a lentiviral vector delivering sgRNA, SpCas9 and coexpressing eGFP (Addgene #57818) according to author instructions (Heckl et al., 2014).

Techniques: Expressing, Alternative Splicing, Control, Modification, Microarray

Journal: eneuro

Article Title: Amyloid Precursor Protein (APP) Controls the Expression of the Transcriptional Activator Neuronal PAS Domain Protein 4 (NPAS4) and Synaptic GABA Release

doi: 10.1523/eneuro.0322-19.2020

Figure Lengend Snippet: Figure 2. APP metabolites regulate NPAS4 expression. A, Schematic representation of APP, its fragments, the AICD-HA construct along with and the localization of the epitopes recognized by the different antibodies used. Western blot analysis of AICD-HA ex- pression after 3 d of lentiviral infection in cells with control or AICD-HA-expressing vectors. Total cell lysate was analyzed with anti- HA antibody. B, Quantification by qPCR of NPAS4 mRNA in neurons APP/ at DIV7 infected with lentiviral vector expressing AICD-HA (n = 6, N = 3). Results are expressed as percentage of control (Ct; mean6 SEM); pp = 0.0291, Student’s t test. C, Medium of sAPPa-treated APP1/1 or APP/ neurons was subjected to Western blot analysis using anti-human APP antibody (clone W0- 2) to detect the exogenous human sAPPa (h sAPPa) and an anti-mouse APP antibody (clone 22C11) to detect both endogenous and exogenous sAPPa (h 1 m sAPPa). Medium was collected after 16 h of treatment. D, Quantification by qPCR of NPAS4 mRNA level in APP1/1 (n = 8, N = 4) or in APP/ neurons at DIV7 treated with 20 nM sAPPa for 16 h (n = 6, N = 3). Results are expressed as percentage of control (Ct; mean 6 SEM); ppp = 0.0055, n.s. = non-significant, Student’s t test. Given that primary cultures of cort- ical neurons at DIV7 also contain astrocytes, the astrocytic pattern of NPAS4 expression is described in Extended Data Figure 2-1.

Article Snippet: A lentiviral vector-based approach was also used to deliver the CRISPR-Cas9 system. sgRNAs Oligo2 and Oligo17 were designed using on/off-target score algorithm to target the APP mouse gene (gene ID: 11820), and sgRNA CRISPR-NPAS4 to target the NPAS4 mouse gene (gene ID: 225872). sgRNAs were cloned in a lentiviral vector delivering sgRNA, SpCas9 and coexpressing eGFP (Addgene #57818) according to author instructions (Heckl et al., 2014).

Techniques: Expressing, Construct, Western Blot, Infection, Control, Plasmid Preparation

Journal: eneuro

Article Title: Amyloid Precursor Protein (APP) Controls the Expression of the Transcriptional Activator Neuronal PAS Domain Protein 4 (NPAS4) and Synaptic GABA Release

doi: 10.1523/eneuro.0322-19.2020

Figure Lengend Snippet: Figure 3. Decreased NPAS4 expression in APP-silenced primary neurons. APP was knock-down by CRISPR-Cas9 approach in primary neurons cultures. The infectivity and toxicity of lentiviral CRISPR-Cas9 vectors are detailed in Extended Data Figure 3-1.

Article Snippet: A lentiviral vector-based approach was also used to deliver the CRISPR-Cas9 system. sgRNAs Oligo2 and Oligo17 were designed using on/off-target score algorithm to target the APP mouse gene (gene ID: 11820), and sgRNA CRISPR-NPAS4 to target the NPAS4 mouse gene (gene ID: 225872). sgRNAs were cloned in a lentiviral vector delivering sgRNA, SpCas9 and coexpressing eGFP (Addgene #57818) according to author instructions (Heckl et al., 2014).

Techniques: Expressing, Knockdown, CRISPR, Infection

Journal: eneuro

Article Title: Amyloid Precursor Protein (APP) Controls the Expression of the Transcriptional Activator Neuronal PAS Domain Protein 4 (NPAS4) and Synaptic GABA Release

doi: 10.1523/eneuro.0322-19.2020

Figure Lengend Snippet: Figure 7. NPAS4 silencing by CRISPR-Cas9 mimics cell phenotype observed in APP-deficient neurons. Changes on inhibitory (GABA) synapses was analyzed after NPAS4 silencing A, left panel, Cortical neurons infected with CRISPR-Cas9 lentivirus targeting NPAS4 gene (CRISPR-NPAS4) show reduced NPAS4 levels as measured by Western blotting (high exposure). Same experiments were conducted after membrane depolarization with 50 mM KCl. NPAS4 accumulations was detectable by Western blotting at low exposure. Viruses with- out sgRNA were used as controls (Ct). Right panel, Quantification of NPAS4 protein level after 2, 3, and 4 h of KCl depolarization. Results (mean 6 SEM) are expressed as percentage of non-treated controls Ct (N =2); pppp, 0.0001 Student’s t test. B, Cortical neurons in- fected with CRISPR-NPAS4 lentiviruses at DIV1 were immunostained against MAP2 and GAD65 at DIV7. Quantification of GAD65 signal was normalized to the number of cells (five fields per coverslip, two coverslips for each genotype in two independent experiments (N=2). Results (mean 6 SEM) are given as percentage of control (Ct). Scale bar: 200 mm; ppp=0.0024. Mann–Whitney test. C, Quantification of GABA in culture medium at DIV7 of infected control neurons (Ct) and CRISPR-NPAS4-infected neurons. Results (mean 6 SEM) are ex- pressed as percentage of Ct (n=5, N=2); pp=0.0146, Student’s t test. D, Neurons harvested at DIV7 and cell extracts analyzed by Western blotting for GABARa1, GAD65, and GADPH expression. Quantification of GABARa1 and GAD65 were normalized to GAPDH expression. Results (mean 6 SEM) are expressed as percentage of Ct (n=8, N =3); pp=0.049, #p=0.0247, Student’s t test.

Article Snippet: A lentiviral vector-based approach was also used to deliver the CRISPR-Cas9 system. sgRNAs Oligo2 and Oligo17 were designed using on/off-target score algorithm to target the APP mouse gene (gene ID: 11820), and sgRNA CRISPR-NPAS4 to target the NPAS4 mouse gene (gene ID: 225872). sgRNAs were cloned in a lentiviral vector delivering sgRNA, SpCas9 and coexpressing eGFP (Addgene #57818) according to author instructions (Heckl et al., 2014).

Techniques: CRISPR, Infection, Western Blot, Membrane, Control, MANN-WHITNEY, Expressing