human synapsin1 promoter  (Addgene inc)

Journal: iScience

Article Title: TAOK2 controls synaptic plasticity and anxiety via ERK and calcium signaling

doi: 10.1016/j.isci.2025.113712

Figure Lengend Snippet: MAPK and calcium signaling is reduced in Taok2 cKO neurons (A–D) Luciferase assay using EGR1p (A), SARE- (B), FOSBp- (C), and CRE- (D) sensor-detected MAPK (A–C) and calcium (B, D)-dependent signaling activity in primary cortical neurons. Neurons were treated with AMPA (1 μM), BDNF (10 ng/mL), or bicuculline (BIC, 1 μM) on day in vitro 12 (DIV12) for 4 h. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; n.s., not significant; Student’s t test (two-sided). (E) Imaging of control and Taok2 cKO primary cortical neurons infected with AVVs expressing the synapsin1-driven GCaMP6f calcium sensor. Images were taken before and after stimulating cells with 25 mM KCl. (F) Quantification of cellular GCaMP6f responses to 25 mM KCl. Intensities from responding cells were normalized to the background and pooled for analysis. Gray arrowheads on the x axis represent the time points at which images were taken before and after stimulation as shown in (E). The line represents the mean, and the shaded area represents the SEM; n = 30 responding cells from two independent cultures. ∗∗∗ p < 0.001 and mixed ANOVA. (G) Western blots indicate reduced phosphorylated Mek1/2 (p-Mek1/2) and Erk1/2 (p-Erk1/2) in primary mouse cortical neurons. Neurons were treated with AMPA (1 μM) on DIV12 for 4 h. (H and I) Quantification of western blot data shown in (G). Data of p-Erk1/2 are relative to total Erk1/2 (H), and data of p-Mek1/2 are relative to total Mek1/2 (I). Values are presented as mean ± SD. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; Wilcoxon rank-sum test (two-sided).

Article Snippet: On DIV6, half the medium was changed and the neurons were infected with AAVs for expression of mRuby2-P2A-GCaMP6f under the control of the human synapsin1 promoter (hSyn1p) (Addgene #50943) at an MOI of 1000.

Techniques: Luciferase, Activity Assay, In Vitro, Imaging, Control, Infection, Expressing, Western Blot

Journal: Neural Regeneration Research

Article Title: The Citron homology domain of MAP4Ks improves outcomes of traumatic brain injury

doi: 10.4103/NRR.NRR-D-24-00113

Figure Lengend Snippet: CNH in neurons exerts neuroprotection. (A) Experimental design to examine CNH function in astrocytes. Gene expression is controlled by the astrocyte-specific human GFAP ( hGFAP ) promoter. (B) Representative images showing GFAP + cells surrounding the cortical lesion. Scale bar: 50 µm. (C) Quantification of fluorescence intensity of GFAP (mean ± SEM; n = 5 mice per group; P = 0.1767). (D) Representative images showing AT8 surrounding the cortical lesion. Scale bar: 50 µm. (E) Quantification of AT8 fluorescence intensity (mean ± SEM; n = 5 mice per group; P = 0.7153). (F) Representative images of brain sections showing GFAP + glial scar (yellow outlined). Scale bar: 1 mm. (G) Quantification of GFAP + scar volumes (mean ± SEM; n = 5 mice per group; P = 0.7448). (H) Representative images of brain sections for lesion quantification (yellow outlined). Scale bar: 1 mm. (I) Quantification of cortical volumes post injury (mean ± SEM; n = 5 mice per group; P = 0.9039). (J) Experimental design to examine CNH function in neurons. Gene expression is controlled by the neuron-specific human SYN1 ( hSYN1 ) promoter. (K) Representative images showing GFAP + cells surrounding the cortical lesion. Scale bar: 50 µm. (L) Quantification of fluorescence intensity of GFAP (mean ± SEM; n = 6 mice per group; P = 0.0007). (M) Representative images showing immunostaining of AT8 surrounding the cortical lesion. Scale bar: 50 µm. (N) Quantification of AT8 fluorescence intensity (mean ± SEM; n = 6 mice per group; P = 0.0152). (O) Representative images of brain sections showing GFAP + glial scar (yellow outlined). Scale bar: 1 mm. (P) Quantification of GFAP + scar volumes (mean ± SEM; n = 6 mice per group; P = 0.0042). (Q) Representative images of brain sections for lesion quantifications (yellow outlined). Scale bar: 1 mm. (R) Quantification of cortical volumes post injury (mean ± SEM; n = 6 mice per group; P = 0.0227). AAV: Adeno-associated virus; Br: bregma; CCI: controlled cortical impact; CNH: Citron homology domain; dpi: days post-injury; GFAP: glial acidic fibrillary acidic protein; IHC: immunohistochemistry.

Article Snippet: The following plasmids were obtained from Addgene (Watertown, MA, USA): pAd-deltaF6 (#112867), pUCmini-iCAP-PHP.eB (#103005), pAAV2/5 (#104964), pAAV2/9 (#112865), and pAAV-CAG-GFP (#37825). pAAV-hGFAP-GFP was previously described (Wang et al., 2021). pAAV-hSYN1-GFP was made by replacing the hGFAP promoter with the hSYN1 promoter (from Addgene #50457). pAAV-CAG-GFP-CNH , pAAV-CAG-BioID2-CNH , pAAV-hGFAP-CNH , and pAAV-hSYN1-CNH were then constructed through PCR-based subcloning into pAAV-CAG-GFP , pAAV-hGFAP-GFP and pAAV-hSYN1-GFP , respectively.

Techniques: Gene Expression, Fluorescence, Immunostaining, Virus, Immunohistochemistry