Evaluation of protective effects of HYP9 and compound 64402 in conditions of amyloid synaptotoxicity. (A) Representative confocal images of day 14 of in vitro cultivation hippocampal neurons transfected with tdTomato plasmid. Images for control (CTRL) cultures and cultures exposed to oligomeric Aβ42 are shown as indicated. HPF was added at the concentration of 300 nM, HYP9 was tested at concentrations of 100 nM and 1 µM as indicated, compound 64402 was tested at the 1 µM concentration. (B) The average percentages of mushroom spines (%MS) in each experimental condition are present as mean ± S.D. (n ≥ 16 neurons for each group). Untreated hippocampal cultures (CTRL) are shown as the filled bar. Open bars correspond to hippocampal cultures that were treated with oligomeric Aβ42. *P < 0.05; ***P < 0.0005 by one-way ANOVA, following Dunn-Sidak post hoc test.

Evaluation of protective effects of compound 50741 in conditions of amyloid synaptotoxicity. (A) Representative confocal images of day 14 of in vitro cultivation hippocampal neurons transfected with tdTomato plasmid. Images for control (CTRL) cultures and cultures exposed to oligomeric Aβ42 are shown as indicated. HPF was added at the concentration of 30 nM, compound 50741 was tested at concentrations of 1, 10, 30, and 100 nM as indicated. Scale bar, 10 µm. (B) The average percentages of mushroom spines (%MS) in each experimental condition are present as mean ± S.D. (n = 22–32 neurons for each group). Untreated hippocampal cultures (CTRL) are shown as filled bars. Open bars correspond to hippocampal cultures that were treated with oligomeric Aβ42. **P < 0.005; ***P < 0.0005 by two-way ANOVA with Dunn-Sidak post hoc test.

Evaluation of the protective effects of compound 51164 in conditions of amyloid synaptotoxicity. (A) Representative confocal images of day 14 of in vitro cultivation hippocampal neurons transfected with tdTomato plasmid. Images for control (CTRL) cultures and cultures exposed to oligomeric Aβ42 (Aβ) are shown as indicated. HPF was added at the concentration of 30 nM, compound 51164 was tested at concentrations of 1, 10, 30, and 100 nM as indicated. Scale bar, 10 µm. (B) The average percentages of mushroom spines (%MS) in each experimental condition are present as mean ± S.D. (n = 22–32 neurons for each group). Untreated hippocampal cultures (CTRL) are shown as filled bars. Open bars correspond to hippocampal cultures that were treated with oligomeric Aβ42. *P < 0.05; ***P < 0.0005 by two-way ANOVA with Dunn-Sidak post hoc test.

Compound 51164 acts as a positive modulator of TRPC6 channels. (A) Time course of Fura-2 fluorescence Ca²⁺ signals (340/380 ratio) is shown for HEK293 cells transfected with EGFP + TRPC6 plasmids. Traces from individual cells are shown as thin gray lines, and average traces are shown as thick black lines. Cells were incubated in aCSF medium containing 2 mM Ca²⁺. The time of addition of 10 µM HPF or 30 µM of compound 51164 is indicated by black bars above the Fura-2 traces. (B) Average amplitude of Ca²⁺ influx peak is shown as the change in 340/380 Fura-2 ratio signals for the cells exposed to HPF or compound 51164. The results are presented as mean ± S.D. (n = 40 cells). ***P < 0.0001 by two-way ANOVA with Tukey’s post hoc test. (C) Time course of Fura-2 fluorescence Ca²⁺ signals (340/380 ratio) is shown for HEK293 cells transfected with EGFP (GFP) or EGFP + TRPC6 (TRPC6) plasmids as indicated. Traces from individual cells are shown as thin gray lines, and average traces are shown as thick black lines. Cells were moved to modified aCSF medium containing 0.1 mM Ca²⁺ for 2 minutes, and then returned to the medium containing 2 mM Ca²⁺ with the addition of 50 µM of OAG. The time of addition of 30 µM of compound 51164 is indicated by black bars above the Fura-2 traces. (D) Average amplitude of Ca²⁺ influx peak is shown as the change in 340/380 Fura-2 ratio signals for each group of cells tested in experiments shown in (C). The results are presented as mean ± S.D. (n = 40 cells). ***P < 0.0001 by two-way ANOVA with Tukey’s post hoc test.

Compound 51164 rescues Aβ₄₂-supressed nSOCE in hippocampal postsynaptic dendritic spines. (A–I) The time course of GCaMP5.3 fluorescence signal changes in the spines of hippocampal neurons at day 14 of in vitro cultivation. Neurons were preincubated in Ca²⁺ free media, and 10 mM Ca²⁺ was added as indicated by the black bar above the traces. The experiments were performed in the presence of a drug cocktail of Ca²⁺ inhibitors as described in Materials and Methods. The presence of 300 nM HPF or 300 nM of compound 51164 is indicated above the GCaMP5.3 traces. For each experimental group, individual spine (thin gray lines) and average (thick black lines) fluorescence traces are shown. Results with control (CTRL) neurons are shown in (A–C). Results with neurons preincubated with Aβ42 oligomers (Aβ) are shown in (D–F) as indicated. Results depicted in (A–F) were obtained in the presence of control shRNA (CTRLsh). Results with neurons transfected with shTRPC6 (T6sh) plasmids are shown in (G–I). (J) The average nSOCE spine peak amplitude is shown for each group of cells depicted in (A–I). The F/F₀ changes in GCaMP5.3 fluorescence are presented as mean ± S.D. (n = 20 spines),*P < 0.05; ***P < 0.0005 by Kruskal-Wallis ANOVA.