cdc42  (Cytoskeleton Inc)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Intersectin1/cdc42 signaling regulates methamphetamine-induced neuronal remodeling in the hippocampus

doi: 10.1007/s00018-025-05890-8

Figure Lengend Snippet: Meth Induces Spatially Restricted Dysregulation of Rho GTPase Signaling in Neurites. A : Schematic representation of primary hippocampal neurons cultured on a modified Boyden chamber system for 14DIV, to separate soma and neurite fractions for RNA sequencing. B : Representative images of upper (soma); lower (neurites) and side compartments of membrane (green: phalloidin, blue: DAPI). C : Dot plot of GSEA (molecular function) for neurites in control vs Meth-exposed neurons. D : “GTPase cycle” heatmap of the relative expression values (z-score of each gene across sample) found in neurites from control vs Meth-exposed neurons. Each column represents individual experiments, which consist of pools from four independent primary hippocampal neuronal cultures. E : Number of repeated interactions established in the first shell of interactors of each term found in neurites involved in “GTPase cycle”. F : Representative images of primary hippocampal neurons transfected at 11DIV with the Raichu-cdc42 probe and treated, at 13DIV, with PBS (Control) or 100 µM Meth for 5, 10, 15 or 30 min (scale bar: 5 µm). G : Graph displays the FRET/Donor ratio in each dendritic spine, normalized to the area of the spine. Symbols represent the mean of each independent cell culture, and the violin plots the variability of all dendritic spines quantified. ** p < 0.01 **** p < 0.0001 (n = 210–248 dendritic spines from 3 independent cultures) (linear mixed analysis with the culture fitted as random effect followed by a Tukey–Kramer correction). H : Schematic representation of experimental timeline of hippocampal synaptosome collection. Mice received a binge administration of Meth and were sacrificed 15 min after the last injection. Hippocampal synaptosomes were isolated and a pull-down assay for GTP-bound cdc42 was performed. I : Graph displays (mean ± SEM) GTP-bound cdc42/Total cdc42 ratio in synaptosomes of the hippocampus of mice 15 min after treatment with Saline or Meth (4 × 5mg/kg, 2h interval), normalized to control animals *p < 0.05 (unpaired t-test) (n = 5–6 animals per group). J : Graph displays (mean ± SEM) Itsn1/GAPDH ratio in synaptosomes of the hippocampus of mice 15 min after treatment with Saline or Meth (4 × 5mg/kg, 2h interval), normalized to control animals (unpaired t-test revealing no significant differences) (n = 5 animals per group). K : Graph displays (mean ± SEM) N-WASP/GAPDH ratio in synaptosomes of the hippocampus of mice 15 min after treatment with Saline or Meth (4 × 5mg/kg, 2h interval), normalized to control animals (unpaired t-test revealing no significant differences) (n = 5 animals per group). L : Graph displays (mean ± SEM) pN-WASP/N-WASP ratio in synaptosomes of the hippocampus of mice 15 min after treatment with Saline or Meth (4 × 5mg/kg, 2h interval), normalized to control animals **p < 0.01 (unpaired t-test) (n = 4–5 animals per group). M : Graph displays (mean ± SEM) Arp3/GAPDH ratio in synaptosomes of the hippocampus of mice 15 min after treatment with Saline or Meth (4 × 5mg/kg, 2h interval), normalized to control animals **p < 0.01 (unpaired t-test) (n = 5 animals per group). N : Itsn1 interacts with cdc42 and N-WASP, which leads to actin polymerization via Arp2/3 complex

Article Snippet: Cdc42 activity was measured using the Pull-down Activation Assay Biochem Kit (#BK034, Cytoskeleton), according to the manufacturer’s instructions.

Techniques: Cell Culture, Modification, RNA Sequencing, Membrane, Control, Expressing, Transfection, Injection, Isolation, Pull Down Assay, Saline

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Intersectin1/cdc42 signaling regulates methamphetamine-induced neuronal remodeling in the hippocampus

doi: 10.1007/s00018-025-05890-8

Figure Lengend Snippet: Cdc42 inhibition prevented Meth-induced neuronal remodeling in vitro. A : Representative images of primary hippocampal neurons transfected with GFP (Control) or miRNA targeting Itsn1, that expresses EmGFP as a reporter, and treated with PBS (Control) or 100 µM Meth treatment for 24h (scale bar: 20 µm). Graphs display (mean ± SEM) B : total neurite length * p < 0.05 ** p < 0.01 (linear mixed analysis with the culture fitted as random effect followed by a Tukey–Kramer correction) and C : ramification (sholl) analysis (n = 21–24 neurons from 3 independent cultures) (two-way ANOVA revealing a significant effect for the distance to the cell body ( p < 0.0001), treatment ( p < 0.0001), and interaction between factors ( p < 0.0001)). D : Representative images of neurons transfected with GFP (Control) or miRNA targeting Itsn1 and treated with PBS (Control) or 100 µM Meth treatment for 24h (scale bar: 5 µm). Graphs display (mean ± SEM) E : total dendritic spine density, F : filopodia spine density, G : thin spine density, H : stubby spine density and I : mushroom spine density ** p < 0.01 **** p < 0.0001 (linear mixed analysis with the culture fitted as random effect followed by a Tukey–Kramer correction revealing no significant differences) (n = 20–27 dendrite segments from 3 independent cultures). J : Representative images of primary hippocampal neurons transfected with cdc42 DN and treated with PBS (Control) or 100 µM Meth treatment for 24h (scale bar: 20 µm). Graphs display (mean ± SEM) K : total neurite length (linear mixed analysis with the culture fitted as random effect followed by a Tukey–Kramer correction revealing no significant differences) and L : ramification (sholl) analysis (n = 17–21 neurons from 3 independent cultures) (two-way ANOVA revealing significant effects for treatment (p < 0.0001) and distance to cell body ( p < 0.0001), but no interaction between factors). M : Representative images of primary hippocampal neurons transfected with cdc42 DN and treated with PBS (Control) or 100 µM Meth treatment for 24h (scale bar: 5 µm). Graphs display (mean ± SEM) N: total dendritic spine density, O : filopodia spine density, P : thin spine density, Q : stubby spine density and R : mushroom spine density (linear mixed analysis with the culture fitted as random effect followed by a Tukey–Kramer correction revealing no significant differences) (n = 18–21 dendrite segments from 3 independent cultures). Dash lines represent: Control (grey) and 100 µM Meth (pink) values from Fig. . Each circle represents a hippocampal neuron/dendrite segment and different colors represent different independent cultures

Article Snippet: Cdc42 activity was measured using the Pull-down Activation Assay Biochem Kit (#BK034, Cytoskeleton), according to the manufacturer’s instructions.

Techniques: Inhibition, In Vitro, Transfection, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Intersectin1/cdc42 signaling regulates methamphetamine-induced neuronal remodeling in the hippocampus

doi: 10.1007/s00018-025-05890-8

Figure Lengend Snippet: Binge Meth Administration Drives Structural and Functional Plasticity in the Hippocampus via cdc42. A : Schematic representation of experimental timeline. Adult male cdc42 fl/fl mice were intravenously injected with an AAV_PHP.eB for tdTomato transduction in the CNS. One week later, mice received a stereotactic surgery for injection of hSyn-GFP or hSyn-Cre-GFP in the hippocampus. Twenty-eight days later, mice received a binge administration of 5 mg/kg Meth, i.p. four times, with two-hours interval between each injection or saline (control). Twenty-four hours after the first administration, mice were sacrificed and the morphology of neurons in the CA1 region of the hippocampus analyzed. B : Schematic representation of stereotaxically injected AAVs. C : Representative images of hippocampal infection (scale bar: 200 µm) (green: GTP, red: tdTomato). Graph display (mean ± SEM) D : cdc42 levels normalized to GAPDH p ** < 0.001 (unpaired t-test) (n = 2–3 mice/group). Graphs display (mean ± SEM) E : total neurite length of neurons of the CA1 region ** p < 0.01 **** p < 0.0001 (linear mixed analysis with the animal fitted as a random effect model followed by a Tukey–Kramer correction) and F : ramification (sholl) analysis **** p < 0.0001 (two-way ANOVA revealing significant effects for treatment ( p < 0.0001) and distance to cell body ( p < 0.0001), with interaction between factors ( p < 0.0001) (n = 5–11 neurons/animal from 3 mice per group). G : Representative tracings of CA1 neurons. H : Representative images of dendritic spines (scale bar: 5 µm). Graphs display (mean ± SEM) I : total dendritic spine density, J : mushroom spine density, K : stubby spine density, L : thin spine density and M : filopodia spine density * p < 0.05 ** p < 0.01 (linear mixed analysis with the animal fitted as a random effect followed by a Tukey–Kramer correction). Each circle represents each hippocampal neuron/dendrite segment and each color represents each mice. N : Summary plot of normalized fEPSP slope measurements recorded in the CA1 region of the hippocampus (mean ± SEM) of cdc42 fl/fl ; hSyn-GFP saline (blue round symbols), cdc42 fl/fl ; hSyn-GFP Meth (yellow square symbols) or cdc42. fl/fl ; hSyn-Cre-GFP Meth (green diamond symbols) treated mice after LTD induction with a low frequency burst stimulation. Slice viability was assessed by recording a second independent pathway that did not show a significant decrease throughout the recording. Right: average fEPSPs at baseline (solid line), T1 (dashed line) and T2 (dotted line). Scale bar: 1 mv, 10 ms. O : Summary plot showing the percentage of LTD decay for the time window at T1 and T2 (two-way ANOVA with two-stage step-up method of Benjamini, Krieger and Yekutiel for multiple comparisons revealing no significant differences) (n = 11–12 slices from 4–5 mice per group)

Article Snippet: Cdc42 activity was measured using the Pull-down Activation Assay Biochem Kit (#BK034, Cytoskeleton), according to the manufacturer’s instructions.

Techniques: Functional Assay, Injection, Transduction, Saline, Control, Infection