Inhibition of Calcineurin with FK506 Reduces Tau Levels and Attenuates Synaptic Impairment Driven by Tau Oligomers in the Hippocampus of Male Mouse Models

Abstract

Alzheimer's disease (AD) is the most common age-associated neurodegenerative disorder, characterized by progressive cognitive decline, memory impairment, and structural brain changes, primarily involving Aβ plaques and neurofibrillary tangles of hyperphosphorylated tau protein. Recent research highlights the significance of smaller Aβ and Tau oligomeric aggregates (AβO and TauO, respectively) in synaptic dysfunction and disease progression. Calcineurin (CaN), a key calcium/calmodulin-dependent player in regulating synaptic function in the central nervous system (CNS) is implicated in mediating detrimental effects of AβO on synapses and memory function in AD. This study aims to investigate the specific impact of CaN on both exogenous and endogenous TauO through the acute and chronic inhibition of CaN. We previously demonstrated the protective effect against AD of the immunosuppressant CaN inhibitor, FK506, but its influence on TauO remains unclear. In this study, we explored the short-term effects of acute CaN inhibition on TauO phosphorylation and TauO-induced memory deficits and synaptic dysfunction. Mice received FK506 post-TauO intracerebroventricular injection and TauO levels and phosphorylation were assessed, examining their impact on CaN and GSK-3β. The study investigated FK506 preventive/reversal effects on TauO-induced clustering of CaN and GSK-3β. Memory and synaptic function in TauO-injected mice were evaluated with/without FK506. Chronic FK506 treatment in 3xTgAD mice explored its influence on CaN, Aβ, and Tau levels. This study underscores the significant influence of CaN inhibition on TauO and associated AD pathology, suggesting therapeutic potential in targeting CaN for addressing various aspects of AD onset and progression. These findings provide valuable insights for potential interventions in AD, emphasizing the need for further exploration of CaN-targeted strategies.

Keywords: Alzheimer’s disease; FK506; Tau; calcineurin; oligomers.

Figure 1

Graphical representation of experimental design.

Figure 2

Effects of FK506 administration on TauO-induced increased levels/visibility of CaN in the mouse hippocampus. (a) Western blot analysis of CaN protein levels across the groups and relative quantification as function of average of PBS + aCSF mice; (b–d) (Left panels) Representative immunofluorescence images of CaN (red) levels in (b) dentate gyrus (DG), (c) CA1, and (d) CA3, from control mice (PBS + aCSF and FK506 + aCSF), mice ICV injected with TauO (PBS + TauO), and mice ICV injected with TauO and treated with FK506 (FK506 + TauO)). Original magnification (60×), scale bar (30 µm) and relative quantitative analyses (right) of the fluorescence intensity across all experimental groups. White arrows and white arrowheads indicate CaN distribution across the hippocampal regions analyzed. (d) Data represent the mean ± SEM; biological replicates n = 4 per group; one-way ANOVA with Tukey’s post hoc corrections for multiple comparison; ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 3

Effects of FK506 administration on hippocampal TauO levels and phosphorylation. Representative immunofluorescence images and relative quantification analyses of Tau (green), p-Tau (AT8-red), and their ratio in (a–d) dentate gyrus (DG), (e–h) CA1, and (i–l) CA3 of control mice (PBS + aCSF and FK506 + aCSF), mice ICV injected with TauO (PBS + TauO), and mice ICV injected with TauO and treated with Fk506 (FK506 + TauO). Original magnification (60×), scale bar (30 µm). Data represent the mean ± SEM; biological replicates n = 4 per group; one-way ANOVA with Tukey’s post hoc corrections for multiple comparison; * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 4

Longitudinal in vivo imaging of mice ICV injected with labeled TauO and treated with FK506. (a) Graphical description of the experimental design; (b) quantification analysis of the fluorescence intensity within the region of interest (ROI) in PBS + TauO and FK506 + TauO mice. (c) Graphical description of the experimental design with the collection of the brains as final step; (d) quantification analysis of the fluorescence intensity within the region of interest (ROI) in PBS + TauO and FK506 + TauO mouse brains. The fluorescence threshold, established by PBS + aCSF and FK506 + aCSF baseline signals, guided the analysis of ROI around the fluorescent signals. Data represent the mean ± SEM; t biological replicates n = 10 per group; one-way ANOVA with Šídák’s post hoc corrections for multiple comparison; * p < 0.05 and *** p < 0.001.

Figure 5

TauO impairs hippocampal-dependent memory function in a CaN-dependent fashion. (a) Graphical representation of novel object recognition (NOR) behavioral test performed on PBS + aCSF, PBS + TauO, and FK506 + TauO mice; time spent with old and new objects calculated as per object discrimination index (ODI) during (b) training, (c) 2 h recall, and (d) 24 h recall behavioral test phases. Data represent the mean ± SD; biological replicates n = 6 per group; one-way ANOVA with Dunn’s test for multiple comparison; * p < 0.05.

Figure 6

TauO suppression of long-term potentiation in a CaN-dependent fashion. (a) Graphical representation of experimental design; (b–d) high-frequency stimulation long-term potentiation in 6 control mice (PBS + aCSF), 6 mice ICV injected with TauO (PBS + TauO), and 8 mice ICV injected with TauO and treated with FK506 (FK506 + TauO). Data represent the mean ± SD; n = 6–8 animals were used for the treatment of the slices as shown in the schematic. The dots represent the total number of slices that were treated. n = 19 for control; n = 13 for TauO, and n = 15 for TauO + FK506. One-way ANOVA with Dunn’s test for multiple comparison; ** p < 0.01, and **** p < 0.0001.

Figure 7

Impact of chronic FK506 on AD neuropathology in 3xTgAD mice. (a–c) Representative immunofluorescence images (upper panels) and relative quantification analyses (lower panels) of Aβ (green) and Tau (red) in (a) dentate gyrus (DG), (b) CA1, and (c) CA3 of control and FK506-treated 3xTgAD mice; (d,e) representative Western blot analyses of (d) AβO and (e) TauO and relative quantifications in the hippocampus of control and FK506-treated 3xTgAD mice. Original magnification (60×), scale bar (30 µm). Data represent the mean ± SD; biological replicates n = 3 (3xTgAD Ctrl) and n = 4 (3xTgAD + FK506); one-tailed t-test; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.