Calsenilin, a Presenilin Interactor, Regulates RhoA Signaling and Neurite Outgrowth

Abstract

Calsenilin modulates A-type potassium channels, regulates presenilin-mediated γ-secretase activity, and represses prodynorphin and c-fos genes expression. RhoA is involved in various cellular functions including proliferation, differentiation, migration, transcription, and regulation of the actin cytoskeleton. Although recent studies demonstrate that calsenilin can directly interact with RhoA and that RhoA inactivation is essential for neuritogenesis, it is uncertain whether there is a link between calsenilin and RhoA-regulated neuritogenesis. Here, we investigated the role of calsenilin in RhoA-regulated neuritogenesis using in vitro and in vivo systems. We found that calsenilin induced RhoA inactivation, which accompanied RhoA phosphorylation and the reduced phosphorylation levels of LIM kinase (LIMK) and cofilin. Interestingly, PC12 cells overexpressing either full-length (FL) or the caspase 3-derived C-terminal fragment (CTF) of calsenilin significantly inactivated RhoA through its interaction with RhoA and p190 Rho GTPase-activating protein (p190RhoGAP). In addition, cells expressing FL and the CTF of calsenilin had increased neurite outgrowth compared to cells expressing the N-terminal fragment (NTF) of calsenilin or vector alone. Moreover, Tat-C3 and Y27632 treatment significantly increased the percentage of neurite-bearing cells, neurite length, and the number of neurites in cells. Finally, calsenilin deficiency in the brains of calsenilin-knockout mice significantly interfered with RhoA inactivation. These findings suggest that calsenilin contributes to neuritogenesis through RhoA inactivation.

Keywords: RhoA; calsenilin; neurite outgrowth; p190RhoGAP.

Figure 1

Calsenilin interacts with RhoA. (A) PC12 cells stably expressing either vector (VEC) or calsenilin (Cal) were immunoprecipitated with an anti-RhoA antibody and then analyzed by Western blotting with anti-calsenilin, and anti-RhoA antibodies; (B) Total cell lysates from PC12 cells expressing calsenilin were preloaded with GDP or GTPγS, after which the proteins were immunoprecipitated with an anti-RhoA antibody and analyzed by Western blotting using anti-calsenilin and anti-RhoA antibodies. The intensities of the bands in each panel were measured and quantified for each group, and the values are expressed as the mean ± SEM of three independent experiments (n = 3, * p < 0.05; *** p < 0.001); (C) The co-localization of calsenilin with RhoA in HEK293 cells expressing either vector (VEC) or calsenilin (Cal) was assessed by double immunofluorescence staining and confocal microscopy. VEC, pEGFP-N1 vector; Cal, GFP-tagged full-length of human calsenilin; TL, total cell lysates. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. DAPI (blue) was used to counterstain the nuclei. Scale bars, 20 μm.

Figure 2

Calsenilin regulates RhoA inactivation and the RhoA-ROCK-LIMK-cofilin pathway. (A) PC12 cells were transiently transfected with calsenilin in a dose-dependent manner. After 24 h, the cells were lysed, and RhoA-GTP was detected by GST-Rhotekin-RBD pull-down assay; (B) Phosphorylation levels of RhoA, LIMK1/2, and cofilin in PC12 cells expressing calsenilin were analyzed by Western blotting. Statistical differences were determined by one-way ANOVA test with Tukey’s post hoc test (n = 3, * p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 3

Calsenilin regulates RhoA inactivation and the RhoA-ROCK-LIMK-cofilin pathway in the mouse brain:(A) detection of RhoA-GTP levels in the brain of wild-type (WT) and calsenilin knockout (KO) mice; (B) phosphorylation levels of RhoA, LIMK, and cofilin in the whole brain lysates of WT and calsenilin-KO mice; and (C) co-immunoprecipitation of RhoA with calsenilin (anti-4E4) [19] and p190RhoGAP in the whole brain lysates of WT and calsenilin-KO mice. The intensities of the bands in each panel were measured and quantified for each group, and the values are expressed as the mean SEM of three independent experiments (n = 3, * p < 0.05; *** p < 0.001).

Figure 4

Calsenilin-CTF is responsible for the interaction with RhoA: (A) schematic diagram depicting the construction of the pEGFP-N1 vector, calsenilin-FL (aa 1–256), -NTF (aa 1–64), and -CTF (aa 65–256); (B,C) total lysates from HEK293 and PC12 cells expressing pEGFP-N1 vector alone (VEC), full-length calsenilin (FL), or truncated fragments (NTF or CTF) were immunoprecipitated with an anti-RhoA antibody and then analyzed by Western blotting with anti-calsenilin (1F11, amino acid 25-33; 4E4), anti-p190RhoGAP and anti-RhoA antibodies; and (D) the co-localization of calsenilin with RhoA in HEK293 cells expressing VEC, calsenilin-FL, -NTF, or -CTF was determined by double immunofluorescence staining using confocal microscopy. Green, calsenilin; Red, RhoA; Blue, DAPI. Scale bars, 20 μm.

Figure 5

Calsenilin-CTF inactivates RhoA. (A) The expression level of calsenilin was determined by Western blotting with anti-1F11 and anti-4E4 antibodies; (B,C) RhoA-GTP levels and phosphorylation of RhoA and RhoA-mediated signaling proteins in PC12 cells expressing VEC, calsenilin-FL, or truncated fragments (NTF or CTF) were detected and analyzed by Western blotting. The intensities of the bands in each panel were measured and quantified for each group, and the values are expressed as the mean ± SEM of three independent experiments. Statistical differences were determined by one-way ANOVA test with Tukey’s post hoc test (n = 3, * p < 0.05; ** p < 0.01).

Figure 6

Overexpression of calsenilin-FL and -CTF decreased F-actin formation. (A) Immunocytochemical staining for F-actin in HEK293 cells expressing VEC, calsenilin-FL, -NTF, or -CTF. The cells were fixed with 4% PFA and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS). F-actin (green) was stained with Alexa Fluor 555-phalloidin. All pictures are representative of multiple images from three independent experiments. Scale bars, 20 μm. (B) The expression of F-actin/G-actin as assessed by F-actin/G-actin in vivo assay in HEK293 cells expressing VEC, calsenilin-FL, -NTF, or -CTF. The intensities of the bands in each panel were measured and quantified for each group, and the values are expressed as the mean ± SEM of three independent experiments. Statistical differences were determined by one-way ANOVA test with Tukey’s post hoc test (n = 3, * p < 0.05; ** p < 0.01).

Figure 7

Overexpression of calsenilin FL and CTF enhanced neurite outgrowth by decreasing RhoA activity and RhoA-mediated signaling. (A–C) Morphological changes in PC12 cells expressing VEC, calsenilin FL, -NTF, or -CTF treated with 50 ng/mL NGF 2.5S for 72 h. All values are expressed as the mean ± SEM of three independent experiments. Statistical differences were determined by one-way ANOVA test with Tukey’s post hoc test (n = 10–20 cells per treatment group, * p < 0.05; ** p < 0.01); (D–F) The cells were incubated with or without 10 μM Y27632 and 1 μg Tat-C3 for 72 h after NGF treatment. The number of neurites per cell was determined by counting all the processes longer than two cell diameters in length. The changes in neurite outgrowth were measured by using INFINITY analysis software. The data represent the mean ± SEM of three independent experiments. Statistical data were obtained by two-way ANOVA with Bonferroni’s post hoc test (n = 10–20 cells per treatment group, * p < 0.05; ** p < 0.01; *** p < 0.001). Scale bars, 100 μm.

Figure 8

Overexpression of calsenilin-FL and -CTF enhanced RhoA inactivation and neurite outgrowth. In calsenilin-FL and -CTF expressing cells, calsenilin increased the phosphorylation of RhoA, enhancing the interaction between RhoA and p190RhoGAP. This complex led to the inactivation of RhoA and its downstream effector proteins. Subsequently, RhoA inactivation decreased actin polymerization and increased neurite outgrowth. In contrast, depletion of calsenilin prevented RhoA inactivation and neurite outgrowth by interfering with the interaction.