Fig 2: NCAM colocalizes with TRPC1, −4, and −5 in hippocampal and cerebellar neurons. Cultured neurons were subjected to double immunostaining with rabbit NCAM antibody GTX133217 and mouse TRPC1 antibody E-6, TRPC4 antibody N77/15, or TRPC5 antibody N67/15, as well as with mouse PSA antibody 735 and rabbit TRPC1 antibody GTX54876 or rabbit TRPC4 antibody ACC-119. Nuclei were stained with DAPI (blue). (a,b) Representative images are shown for coimmunostaining of TRPC1 (red) and NCAM (green) or of TRPC1 (green) and PSA (red) in cerebellar (a) and hippocampal (b) neurons. Superimposition of red and green stainings shows colocalization in yellow. Scale bar: 20 µm. (c,d) Pearson’s coefficients were determined from 10 immunostained neurons per group and from 2 different cultures. Box plots show the Pearson’s coefficients and indicate an overlap of NCAM or PSA stainings with TRPC stainings in cerebellar (c) and hippocampal (d) neurons.

Fig 3: NCAM regulates the Ca2+ entry via TRPC1, −4, and −5 in transfected CHO cells that express PSA-NCAM. NCAM-expressing CHO 2A10 cells, PSA-NCAM-expressing CHO C6 cells, or NCAM-lacking NCAMneg CHO cells were mock-transfected or transfected with plasmids encoding TRPC1/4 or TRPC1/5 heteromers, loaded with the Ca2+ indicator Fluo-4 AM, treated with or without function-triggering NCAM antibody in the absence or presence of the TRPC inhibitor SKF96365, and subjected to Ca2+ imaging. (a) A representative time course of a Ca2+ response of mock-transfected and TRPC1/4- or TRPC1/5-transfected CHO C6 cells after application of a function-triggering NCAM antibody (arrow; NCAM Ab) is shown (* p < 0.05, ** p < 0.01; mixed effects analysis comparing values from the TRPC1/4- or TRPC1/5-transfected cells with the mock-transfected cells). (b–f) Scatter plots show mean values ± SEM and single values for the peak Fluo-4 AM intensity values of 6–8 cells per treatment for mock-transfected and TRPC1/4- or TRPC1/5-transfected CHO C6 cells (b,e), CHO 2A10 cells (c,f), and NCAM-lacking NCAMneg CHO cells and in the absence (b–f) and presence (e,f) of SKF96365 (SKF) (* p < 0.05, ** p < 0.01 relative to mock-transfected (b) or vehicle-treated (e) cells; one-way ANOVA with Kruskal–Wallis post hoc test (b–d), Mann–Whitney test (e); ns, not significant). (g–i) Lysates of CHO 2A10 and CHO C6 cells were used for immunoprecipitation (IP) with TRPC1 antibody ACC-010 (g), TRPC4 antibody ACC-018 (h), TRPC5 antibody ACC-020 (h), and antibody GTX133217 against the C-terminus of NCAM (NCAM-CT) (i), and nonimmune control antibodies (Ig) (g–i) were used for immunoprecipitation. The lysates (input) and the immunoprecipitates were subjected to Western blot analysis with NCAM antibody GTX133217 (NCAM-CT) (g,h) or TRPC4 antibody ACC-018 (i).

Fig 4: NCAM colocalizes with TRPC1, −4, and −5 predominantly in the plasma membrane of cortical neurons. (a) The scheme illustrates the principle of TIRF microscopy. A glass coverslip (black bar) and an adhering neuron (light blue) with nucleus and ER (ovals) are depicted. Application of laser light (light green; red arrows) at a certain angle leads to total reflection of light at the interfaces of the glass coverslip and the aqueous film between coverslip and adherent cells, generating an evanescent wave, which creates an evanescent field (yellow area). As the energy in this field decreases exponentially with distance of typically 60–100 nm to the interfaces, only fluorophores in this proximity to the coverslip are excited, leaving other fluorophores in the cell not excited. This allows the detection of protein interactions at the plasma membrane and in the ER membranes near the plasma membrane. Shown within the evanescent field are excited fluorophores indicating TRPC/NCAM interactions (red circles), plasma membrane (PM) marker proteins (blue circles), and ER membrane marker proteins (green circles). Outside of this field, light grey, dark grey, and black circles indicate the corresponding nonexcited fluorophores. (b–e) Cultured wild-type cortical neurons were first transfected with plasmids encoding a plasma membrane-associated fusion protein of blue fluorescence protein and the CAAX motif-containing sequence of H-Ras or encoding an ER membrane-associated fusion protein of enhanced green fluorescence protein, the ER-targeting sequence of calreticulin, and the ER retention sequence KDEL. Subsequently, the cells were treated without (d,e) or with the NCAM antibody (d) or EndoN (e) and subjected to proximity ligation with rabbit NCAM antibody GTX133217 and mouse TRPC1 antibody E-6, TRPC4 antibody N77/15, or TRPC5 antibody N67/15 or with mouse PSA antibody 735 and rabbit TRPC1 antibody ACC-010 or TRPC4 antibody ACC-018. The neurons were then analyzed by TIRF microscopy. (b) Representative images show TRPC1/NCAM interactions (red dots) at the cell surface predominantly near the plasma membrane marker protein (blue) (NCAM/TRPC1/PM), but rarely near the ER membrane marker protein (green) (NCAM/TRPC1/ER) (scale bar: 20 µm). (c) Representative images are shown for interactions of TRPCs with NCAM or PSA at the cell surface (scale bar: 20 µm). The numbers of red dots near to the plasma membrane marker (blue) or to the ER marker (green) were counted separately (overlapping dots were not considered) and indicate the interaction of NCAM or PSA with TRPCs in the plasma membrane or in ER membranes near the plasma membrane. (d,e) Scatter plots show mean values ± SEM and single values for the numbers of plasma membrane-associated (PM) and ER-associated (ER) red dots per cell from 3 independent experiments analyzing 10 neurons per group and experiment (*** p < 0.001; Mann–Whitney test comparing PM and ER values).

Fig 5: NCAM-mediated signal transduction and neurite outgrowth depend on TRPC1, −4, and −5. (a,b) Hippocampal neurons were maintained on PLL, NCAM-Fc (NCAM), L1-Fc (L1), or CHL1-Fc (CHL1) and treated without (−) (a) or with control antibody (Ig) (b), TRPC inhibitor SKF96365 (a), or function-blocking TRPC1 antibody T1E3 (b). Total neurite lengths per neuron were measured to determine neurite outgrowth. Mean values ± SEM from 3 independent experiments with duplicates are shown for neurite outgrowth relative to neurons without additives (* p < 0.01; one-way ANOVA with Dunn’s multiple comparison test) or relative to vehicle control (§ p < 0.01; one-way ANOVA with Dunn´s multiple comparison test). (c) Cortical neurons from wild-type (NCAM+/+) (c) and NCAM-deficient (NCAM−/−) (d) mice were treated with vehicle alone or together with NCAM antibody (NCAM Ab) or colominic acid/PSA (CA) in the absence (c,d) or presence of Pico145 (Pico), HC-070 (HC), or M084 (c). Total neurite lengths per neuron were measured to determine neurite outgrowth. Mean values ± SEM from 3 independent experiments with duplicates are shown for neurite outgrowth relative to neurons incubated without additives (* p < 0.05, **** p < 0.0001; one-way ANOVA with Kruskal–Wallis post hoc test). (e,f) Wild-type cortical neurons were treated with NCAM antibody in the absence or presence of HC-070 (HC) Pico145 (Pico) or colominic acid/PSA (CA) and subjected to Western blot analysis with antibodies against total and phosphorylated Erk1/2 and PKC and with a γ-adaptin antibody for loading control. Levels of total and phosphorylated Erk1/2 and PKC were determined by Western blot analysis. Mean values ± SEM are shown for the level of phosphorylated Erk1/2 (e) and PKC (f) relative to total Erk1/2 and PKC levels and relative to the values obtained for NCAM antibody-stimulated neurons in the absence of inhibitors (set to 100%) (* p < 0.05; one-way ANOVA with Kruskal–Wallis post hoc test).