Tau Protein Mediates APP Intracellular Domain (AICD)-Induced Alzheimer’s-Like Pathological Features in Mice

<div><p>Amyloid precursor protein (APP) is cleaved by gamma-secretase to simultaneously generate amyloid beta (Aβ) and APP Intracellular Domain (AICD) peptides. Aβ plays a pivotal role in Alzheimer’s disease (AD) pathogenesis but recent studies suggest that amyloid-independent mechanisms also contribute to the disease. We previously showed that AICD transgenic mice (AICD-Tg) exhibit AD-like features such as tau pathology, aberrant neuronal activity, memory deficits and neurodegeneration in an age-dependent manner. Since AD is a tauopathy and tau has been shown to mediate Aβ–induced toxicity, we examined the role of tau in AICD-induced pathological features. We report that ablating endogenous tau protects AICD-Tg mice from deficits in adult neurogenesis, seizure severity, short-term memory deficits and neurodegeneration. Deletion of tau restored abnormal phosphorylation of NMDA receptors, which is likely to underlie hyperexcitability and associated excitotoxicity in AICD-Tg mice. Conversely, overexpression of wild-type human tau aggravated receptor phosphorylation, impaired adult neurogenesis, memory deficits and neurodegeneration. Our findings show that tau is essential for mediating the deleterious effects of AICD. Since tau also mediates Aβ-induced toxic effects, our findings suggest that tau is a common downstream factor in both amyloid-dependent and–independent pathogenic mechanisms and therefore could be a more effective drug target for therapeutic intervention in AD.</p></div

Tau mediates AICD-induced AD-like phenotypes.

<p>(A, B) AICD activates GSK-3β; this in turn phosphorylates tau, which accumulates in somato-dendritic compartments. Increased somato-dendritic tau results in a number of AD-like features in AICD animals, including phosphorylation of NMDA receptors (NMDAR), which leads to increased susceptibility to excitotoxicity. (C) Increasing the amount of tau by transgenic overexpression of human tau (h-Tau) resulted in increased somato-dendritic tau and elevated phosphorylation of NMDAR. Increased NMDAR phosphorylation led to increased susceptibility to excitotoxic effects and aggravated AD-like pathologies. (D) Loss of tau prevented increased NMDAR-phosphorylation, resulting in normalization/lowering of hyper-excitability, finally reducing/ameliorating several key AD-like features in AICD mice.</p

Deficiency of tau protects against age-dependent neurodegeneration and memory loss measured by deficits in spontaneous alternations in AICD-Tg mice.

<p>A. AICD-Tg mice exhibit decreased working memory as measured by deficits in spontaneous alternations at older ages. At >18 months of age, AICD-Tg mice showed decreased spontaneous alternations (working memory) compared to age-matched wild-type animals. Lack of tau (Tau-/-) was able to protect AICD-Tg mice from such deficits in working memory. B. Normal exploratory behavior for all the genotypes measured in the Y-maze. n = 10–12 mice per group for behavioral experiments (A and B). C. AICD-Tg mice at more than 18 months of age showed neuronal loss as assessed by NeuN staining in the CA3 region. Arrows point to focal loss of neurons. Genetic ablation of tau (Tau-/-) protected against neurodegeneration in AICD-Tg mice. D. Quantification of NeuN-positive nuclei. n = 4 for all groups. *p<0.05, **p<0.01 one way ANOVA (Mean ± SEM). Scale bar = 50 μm. WT = wild-type.</p

Tau overload causes behavioral deficits and maintains neurodegeneration at older ages.

<p>A. hTau animals also showed decreased working memory as measured by deficits in spontaneous alternations compared to age-matched wild-type animals. AICD-Tg mice exhibit decreased working memory at older ages compared to age-matched wild-type animals. B. Evidence of normal exploratory behavior was demonstrated for all the genotypes measured in the Y-maze. n = 10–12 mice per group for behavioral experiments (A and B). C. Old AICD-Tg mice carrying an overload of human tau showed neuronal loss similar to age-matched AICD-Tg mice without human tau as assessed by NeuN staining in the CA3 region. Arrows point to focal loss of neurons. Though not statistically significant, there was a trend toward hTau mice having less neuronal cell bodies in the CA3 compared to age-matched wild-type animals. D. Quantification of NeuN-positive nuclei. n = 5 for all. *p<0.05, **p<0.01, ***p<0.001 one way ANOVA (Mean ± SEM). Scale bar = 50 μm</p

Inhibiting GSK-3β protects against tau pathology and rescues impaired neurogenesis in AICD-Tg mice.

<p>A. Hippocampal brain lysates from 3-month-old wild-type (WT) mice, AICD-Tg mice and AICD-Tg mice fed lithium chow (AICD+Li) were western blotted using AT8, AT180 or Tau-5 to detect phospho- tau or total tau. AICD animals showed increased phospho-tau compared to WT mice, while total tau levels were not significantly different. Inhibiting the tau kinase GSK-3β with lithium treatment for a month decreased the amount of phosphorylated tau in the hippocampus of AICD-Tg mice. B-C. Quantification of western blots for AT8 and AT180 respectively, showed a significant reduction in the amount of phospho-tau in AICD animals after lithium treatment. D. AICD animals showed increased phosphorylation of NMDA receptors. However, Inhibiting GSK-3β normalized this increased phosphorylation of NMDA receptors in AICD-Tg animals such that they were similar to WT animals. E. Quantification of western blots for phospho-NMDAR and NMDAR. F-G. Inhibiting GSK-3β rescued impaired adult hippocampal progenitor cell (HPC) proliferation in AICD-Tg mice. HPC proliferation measured as the number of BrdU-positive cells showed increased proliferation in AICD animals after lithium treatment, which is quantified in (G). **p<0.01, ***p<0.001 one way ANOVA (Mean ± SEM). n = 5–7 mice per group. Scale bar = 50 μm.</p

Lack of tau normalizes increased p-NMDAR levels in AICD animals.

<p>A. Representative immunoblots showing that AICD-Tg mice at 3–4 months of age have increased pY1472-NR2B (pNMDAR) levels to total NMDAR levels compared to age-matched non-transgenic animals. B. Lack of tau normalizes the increased pNMDAR levels in AICD-Tg animals. C. Quantification of pNMDAR/NMDAR levels in wild-type and AICD animals in the presence (Tau +/+) or absence (Tau-/-) of tau. n = 5–6 for each genotype. *p<0.05, **p<0.01, ***p<0.001 one way ANOVA (Mean ± SEM).</p

Tau knockout prevents sensitivity to Kainic acid–mediated stress in AICD-Tg mice.

<p>A. AICD-Tg mice exhibit increased sensitivity towards Kainic Acid (KA)-mediated seizures at 3–4 months of age. Animals were injected with a sub-threshold dose of KA (20 mg/kg) and observed for 60 minutes for seizure–like hyperactivity. Seizures were scored according to a modified Racine scale. AICD-Tg mice began to show significant signs of seizures at 30 min. However, lack of tau (Tau-/-) significantly protected AICD-Tg mice against KA-mediated seizure-like hyperactivity. Tau-/- mice behaved similar to wild-type (WT) littermates. B. The time taken to reach level 4–5 seizures was scored as the latency to reach convulsive seizures. Lack of tau (Tau-/-) had a protective effect on AICD transgenic animals. C. Mean seizure severity scores showed a significant rescue of seizure severity by Tau-/- in AICD animals. n = 4–5 mice per group (A-C). D. Sagittal sections from 4-month-old WT and AICD-Tg mice were stained with NeuN antibody. Arrows show the loss of neurons in the CA3 region of AICD-Tg mice. Deletion of Tau protected AICD-Tg mice from KA-mediated neuronal loss. E. Quantification of NeuN-positive cells shows that Tau-/- makes AICD-Tg mice resistant to KA-mediated neurodegeneration. F. AICD-Tg mice show an increase in neuropeptide-Y (NPY) expression in mossy fiber terminals (arrows) following KA-mediated seizures. Tau deletion decreased NPY expression in mossy fiber terminals following KA injection in AICD-Tg animals. G. Quantification of NPY immunoreactivity normalized to WT animals. *p<0.05, **p<0.01, ***p<0.001, one way ANOVA (all data expressed as Mean ± SEM). n = 5 for all groups (D-G). Scale bar = 50 μm (D) and100 μm (F).</p

Increased somato-dendritic tau accumulation and phosphorylation of NMDAR after hTau overexpression.

<p>A-B Phospho-tau staining with AT8 (A) and AT180 antibody (B) revealed increased somato-dendritic accumulation of phosoho-tau in the cerebral cortex (A) and hippocampal CA1 layer (B) of hTau animals compared to wild-type (WT) and AICD-Tg transgenic mice at 3–4 mo of age. Scale bar = 100 μm C. Overexpression of human tau (hTau) increased pY1472-NMDAR levels in both WT and AICD animals. D. Quantification of pY1472-NMDAR/NMDAR levels in wild-type and AICD animals in the presence of mouse (mTau) or human tau (hTau). n = 5–6 for each genotype. *p<0.05, **p<0.01 one way ANOVA (Mean ± SEM). Sale bar in A,B = 50 μm.</p

Human tau overexpression impaired adult hippocampal progenitor cell (HPC) proliferation.

<p>A. Representative images of BrdU immunostaining in the subgranular zone (SGZ) of the dentate gyrus performed on 3-month-old wild-type and AICD-Tg animals with endogenous mouse tau (mTau) or overexpressed human tau (hTau). B. Quantitative analysis of the total number of BrdU+ cells throughout the entire rostro-caudal extent of the hippocampus in 3-month-old animals revealed a statistically significant decrease in the number of BrdU+ cells in the SGZ of AICD-Tg mice compared to wild-type mice. However, overexpression of human tau (hTau) alone also decreased HPC proliferation in wild-type animals and had a tendency to decrease HPC proliferation in AICD animals. n = 4–7 for each group. C. Representative images of doublecortin (DCX) immunostaining on 3-month-old wild-type and AICD-Tg animals with endogenous mouse tau (mTau) or overexpressed human tau (hTau) revealed a decrease in the number of immature neurons in both WT and AICD animals that overexpress h-Tau. GC: Granule cell layer; H: Hilus. *p<0.05, **p<0.01 one way ANOVA (Mean ± SEM). Scale bar = 100 μm.</p