Targeted Reducing of Tauopathy Alleviates Epileptic Seizures and Spatial Memory Impairment in an Optogenetically Inducible Mouse Model of Epilepsy

Abstract

Intracellular deposition of hyperphosphorylated tau has been reported in the brain of epilepsy patients, but its contribution to epileptic seizures and the association with spatial cognitive functions remain unclear. Here, we found that repeated optogenetic stimulation of the excitatory neurons in ventral hippocampal CA1 subset could induce a controllable epileptic seizure in mice. Simultaneously, the mice showed spatial learning and memory deficits with a prominently elevated total tau and phospho-tau levels in the brain. Importantly, selective facilitating tau degradation by using a novel designed proteolysis-targeting chimera named C4 could effectively ameliorate the epileptic seizures with remarkable restoration of neuronal firing activities and improvement of spatial learning and memory functions. These results confirm that abnormal tau accumulation plays a pivotal role in the epileptic seizures and the epilepsy-associated spatial memory impairments, which provides new molecular target for the therapeutics.

Keywords: cognitive impairment; epilepsy; mouse model; optogenetics; tau hyperphosphorylation.

FIGURE 1

Optogenetic stimulation of ventral hippocampal excitatory neurons induces controllable epileptic seizures. (A) Schematic illustrates the locations of optic fiber and recording electrode. (B) Experimental procedures of virus injection, optic fiber and electrode implantation, optogenetic seizure induction. (C) A representative image showing the AAVs-mediated mCherry expression in the ventral hippocampus. Scale bar, 500 μm. (D) The representative images show typical seizure behavior (MRS stages 2–5) and the latency to generalized seizures (upper). Repeated optogenetic stimulation of vCA1 excitatory neurons induced typical seizure behavior, and the latency to generalized seizures was decreasing during repeated induction that showed seizure threshold declined over time (bottom). (E,F) Optogenetic hyperactivation of vCA1 excitatory neurons (i.e., blue light on) induced epileptiform neuronal activities in the primary motor cortex (M1). The local field potential (LFP) amplitude in M1 significantly increased during the phase of incubation and generalized seizures (shortened as S). The mice injected with AAV-mCherry were tested as non-seizure controls. Repeat-measures one-way ANOVA followed by Tukey multiple-comparisons tests, **p < 0.01, ***p < 0.001, compared with the baseline, n = 8 in each group. (G,H) Optogenetic induction of seizures increased the power spectra density (PSD) of LFP in the phase of incubation and generalized seizures. Repeated-measures one-way ANOVA followed by Tukey multiple-comparisons tests. **p < 0.01, ***p < 0.001, compared with the baseline, n = 8 in each group. Values are presented as the mean ± SEM.

FIGURE 2

Optogenetic induction of repeated seizures impairs spatial learning and memory without causing anxiety-like behaviors or hippocampal neuron loss in mice. (A) Experimental procedures of virus injection, optic fiber implantation, optogenetic seizure induction, and behavioral tests. (B) Repeated seizure induction caused spatial learning deficits shown by the increased escape latency during training phase. Repeated-measures ANOVA followed by Tukey multiple-comparisons tests, *p < 0.05. (C,D) Repeated seizure induction caused spatial memory deficits shown by the decreased number of target quadrant crossings in water maze test (B) and the representative heatmaps (C) show traveling time and the trace. Unpaired t test, *p < 0.05. (E,H) Repeated but controllable seizures did not induce anxiety-like behavior shown by the unchanged central time (D) and traveled distance (E) in open field test, and the increased open arm stay during elevated plus maze test (F). The representative heatmaps (G) show traveling time and the trace. Unpaired t test, *p < 0.05. (I) Repeated but controllable seizures did not induce neuron loss in the granular cell layer (GCL) of dorsal and ventral hippocampus measured by Nissl staining (p > 0.05). Scale bars were as indicated in each panel. n = 6 mice in each group. Unpaired t tests. Values are presented as the mean ± SEM.

FIGURE 3

Repeated seizures upregulate phosphorylated and total tau levels in the hippocampus and cortex of mice. Repeated seizures increased phospho-tau (AT8) and total tau (Tau5) levels in hippocampus and cortex measured by Western blotting (A,B) and immunostaining (C,D). n = 3 mice in each group. Unpaired t-tests, *p < 0.05, **p < 0.01. Scale bars were as indicated in each panel. Values are presented as the mean ± SEM.

FIGURE 4

C4 selectively facilitates tau degradation in the optogenetically inducible mouse model of epilepsy. (A) Experimental procedure of virus injection, seizure induction, C4 administration, and behavioral tests. (B–D) C4 administration diminished the repeated seizure-induced elevation of total tau and phospho-tau levels in hippocampus and cortex measured by immunostaining (B) and Western blotting (C,D). n = 4 mice in each group. One-way ANOVA followed by Tukey multiple-comparisons tests, *p < 0.05. Scale bars were as indicated in each panel. Values are presented as the mean ± SEM.

FIGURE 5

Selectively facilitating tau degradation by C4 alleviates the optogenetics-induced epileptic seizures and the associated spatial memory deficits. (A–C) Blue light on did not change basal PSD in primary motor cortex (M1) at day 1, whereas C4 treatment significantly downregulated the PSD in M1 at day 14 during light-on. n = 3 mice in each group, unpaired t tests, *p < 0.05. (D,E) C4 prolonged the latency of generalized seizures of mice. n = 7–9 mice in each group. Repeated-measures ANOVA followed by Tukey multiple-comparisons tests, *p < 0.05. Unpaired t-tests, **p < 0.01. (F,G) C4 tended to decrease the seizure duration and severity of seizure stages at day 14. Unpaired t tests, *p < 0.05. (H–J) C4 showed limited effect on improving the repeated seizures-induced spatial learning impairments in the water maze training phase. n = 9–10 mice in each group. Repeated-measures ANOVA followed by Tukey multiple-comparisons tests, *p < 0.05, compared with the non-seizure Ctrl group. (I,J) C4 significantly alleviated the seizures-induced spatial memory impairments. One-way ANOVA followed by Tukey multiple-comparisons tests, *p < 0.05. Values are presented as the mean ± SEM.