Journal: bioRxiv

Article Title: Hyperexcitability in Alzheimer’s Disease triggers a compensatory neuroprotective response via TREK1

doi: 10.1101/2025.10.16.682816

Figure Lengend Snippet: a, Representative calcium imaging traces from control and Aβ42o treated neurons showing that Aβ42o increases spontaneous calcium transient frequency. This hyperexcitability is further enhanced by the TREK1 inhibitor spadin and suppressed by the TREK1 activator BL-1249. b, Quantification of calcium event frequency upon treatment with Aβ42o/spadin/BL-1249 (n = 512–1002 cells; ****p < 0.0001, ##p < 0.01, †p < 0.05; one-way ANOVA with Šidák’s test). c, Representative FluoVolt traces measuring membrane potential fluctuations manifest enhanced neuronal activity in the presence of TREK1 inhibitor spadin and suppressed neuronal activity by the TREK1 activator BL-1249 compared to Aβ42o treatment alone. d, Quantification of potential spike frequency upon treatment with Aβ42o/spadin/BL- 1249 (n = 31–105 cells; **p < 0.01, #p < 0.05, ††p < 0.01; one-way ANOVA with Šidák’s test). e, Representative calcium traces from neurons treated with Aβ42o along with scrambled (Sc) siRNA, KCNK2 siRNA, or a KCNK2 overexpression (OE) construct. f, Quantification of calcium event frequency upon knocking down KCNK2 in the presence of Aβ42o treatment (n = 49–67 cells; *p < 0.05; unpaired t-test). g, Quantification of calcium event frequency upon overexpressing KCNK2 in the presence of Aβ42o treatment (n = 37–48 cells; *p < 0.05, #p < 0.05; one-way ANOVA with Šidák’s test). h, Representative patch-clamp recordings of action potentials in control, Aβ42o, and Aβ42o + spadin treated neurons showing exacerbated action potential firing with TREK1 blockade. i, Quantification of action potential frequency following Aβ42o and/or spadin treatment (n = 17 cells; *p < 0.05, #p < 0.05; one-way ANOVA with Šidák’s test). j, Resting membrane potential (RMP) is more depolarized in Aβ42o treated neurons compared to control, and further depolarizes in presence of spadin with Aβ42o (n = 19 cells; ****p < 0.0001, #p < 0.05; one-way ANOVA with Šidák’s test). k, Representative traces showing excitatory postsynaptic current (EPSC) frequency is increased in neurons treated with Aβ42o + spadin compared to Aβ42o alone. l, Quantification of EPSC frequency (n = 19 cells; *p < 0.05, ##p < 0.01; one-way ANOVA with Šidák’s test). m, Quantification of EPSC amplitude (n = 19 cells). n, Representative traces showing Inhibitory postsynaptic current (IPSC) frequency is decreased in neurons treated with Aβ42o+spadin compared to Aβ42o alone. o, Quantification of IPSC frequency (n = 10 cells; **p < 0.01, #p < 0.05; one-way ANOVA with Šidák’s test). p, Quantification of IPSC amplitude (n = 10 cells). q, Representative ex vivo calcium imaging heat map from hippocampal slices of 3xTg mice injected with TREK1 shRNA lentivirus showing elevated calcium activity compared to sc shRNA-injected mice. r, Representative calcium imaging traces demonstrating increased calcium transient frequency following TREK1 knockdown. s, Quantification of calcium event frequency in TREK1 knockdown mice compared to sc shRNA-injected mice (n = 13–27 cells; *p < 0.05; unpaired t-test). Data are presented as mean ± SEM from 3-5 independent cultures.

Article Snippet: For lentiviral-mediated knockdown studies, 1 × 106 IFU of CTCF shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-35125-V) or KCNK2 shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-37181-V) or control shRNA lentiviral particles (Santa Cruz Biotechnology, #sc-108080) were stereotaxically delivered into the hippocampus of 3xTg mice in a total injection volume of 5 μL, and animals were maintained for 15 days post-injection before brain isolation.

Techniques: Imaging, Control, Membrane, Activity Assay, Over Expression, Construct, Patch Clamp, Ex Vivo, Injection, shRNA, Knockdown