Behavioral deficit, oxidative stress, and mitochondrial dysfunction precede tau pathology in P301S transgenic mice

Abstract

Abnormal tau accumulation can lead to the development of neurodegenerative diseases. P301S mice overexpress the human tau mutated gene, resulting in tau hyperphosphorylation and tangle formation. Mice also develop synaptic deficits and microglial activation prior to any neurodegeneration and tangles. Oxidative stress can also affect tauopathy. We studied the role of oxidative stress in relationship to behavioral abnormalities and disease progression in P301S mice at 2, 7, and 10 mo of age. At 7 mo of age, P301S mice had behavioral abnormalities, such as hyperactivity and disinhibition. At the same age, we observed increased carbonyls in P301S mitochondria (∼215 and 55% increase, males/females), and deregulation in the activity and content of mitochondrial enzymes involved in reactive oxygen species formation and energy metabolism, such as citrate synthase (∼19 and ∼5% decrease, males/females), MnSOD (∼16% decrease, males only), cytochrome C (∼19% decrease, females only), and cytochrome C oxidase (∼20% increase, females only). These changes in mitochondria proteome appeared before tau hyperphosphorylation and tangle formation, which were observed at 10 mo and were associated with GSK3β activation. At that age, mitochondria proteome deregulation became more apparent in male P301S mitochondria. The data strongly suggest that oxidative stress and mitochondrial abnormalities appear prior to tau pathology.

Figure 1.

P301S transgenic mice showed hyperactivity and disinhibition. A–F) Total distance traveled (A–C) and rearing frequency (D–F) in the open-field test at 2 mo (A, D), 7 mo (B, E), and 10 mo (C, F). P301S (Tg) mice were hyperactive compared to wild-type (Wt) littermates at 7 and 10 mo. G–L) Frequency of entries in closed and open arms (G–I), as well as percentage of time spent in open arms (J–L), in the elevated plus maze at at 2 mo (G, J), 7 mo (H, K), and 10 mo (I, L). P301S mice were hyperactive and disinhibited compared to wild-type littermates at 7 and 10 mo. Data are expressed as means ± se.

Figure 2.

P301S transgenic mice did not present major cognitive deficits. Acquisition period (A–C) and probe trial (D–F) of the Morris water maze in which latency to reach the hidden platform and percentage of time spent in each quadrant were recorded at 2 mo (A, D), 7 mo (B, E), and 10 mo (C, F). No difference was found between P301S (Tg) mice and wild-type (Wt) littermates in the Morris water maze. However, trend indicated increased latency to reach the platform in P301S mice at 10 mo. Data are expressed as means ± se.

Figure 3.

Tau phosphorylation was augmented in aged P301S transgenic mice. A–C) Western blots of AT8, CP13, and PHF1 in the cortex (A), hippocampus (B), and spinal cord (C) of P301S (Tg) mice at 2, 7, and 10 mo of age. D–L) Quantification of AT8 (D–F), CP13 (G–I), and PHF1 (J–L) levels in the cortex (D, G, J), hippocampus (E, H, K), and spinal cord (F, I, L), using ratio of each antibody to β-actin. P301S mice (10 mo old) had increased AT8, CP13, and PHF1 levels compared to 2- and 7-mo-old P301S mice. Data are expressed as means ± se.

Figure 4.

GSK3β level was augmented in aged P301S transgenic mice. A–C) Western blots of GSK3β at 2 mo (A), 7 mo (B), and 10 mo (C) in P301S (Tg) mice and their wild-type (Wt) littermates. D–F) Quantification of GSK3β level at 2 mo (D), 7 mo (E), and 10 mo (F) using ratio of the protein to β-actin. P301S mice (10 mo) had increased GSK3β levels compared to nontransgenic mice. Data are expressed as means ± se.

Figure 5.

Tau accumulation and tangles were increased with age in P301S transgenic mice. MC1 immunoreactivity in the somatosensory and piriform cortices, the CA1 region of the hippocampus, and the central amygdala of 7-mo-old wild-type (Wt) mice, as well as 2-, 7-, and 10-mo-old P301S (Tg) mice. Scale bars = 250 μm. Higher-magnification photographs were taken in 10-mo-old P301S mice to visualize tangles. MC1 immunoreactivity increased from 2 to 10 mo in P301S mice.

Figure 6.

Oxidative stress was enhanced in the brain of P301S transgenic mice. A) Levels of MDA at 2, 7, and 10 mo. No significant difference was found between P301S (Tg) mice and their wild-type (Wt) littermates. B–D) Western blots of carbonyls (B, C) and quantification of carbonyl level (D, E) at 7 mo (B, D) and 10 mo (C, E). Levels of carbonyls in 7- and 10-mo-old P301S mice were increased in the cortex and compared to nontransgenic mice. F, G) ROS generation with FADH-linked (F) and NADH-linked substrates (G) at 7 mo. No differences were found in ROS generation. St4, state 4; St3, state 3; Rot, rotenone; Ant, antimycin. Data are expressed as means ± se.

Figure 7.

Deficits in the respiratory chain and the tricarboxyl acid cycle were present in P301S transgenic mice. Mitochondrial enzyme activity of CS, CI, SDH, GR, and ACO, and protein expression of cytochrome c, MnSOD, total SOD (SOD), CI, CIII, CIV (COXIV) and ATPase at 7 mo (A, B) and 10 mo (C, D) in male (A, C) and female (B, D) P301S (Tg) mice and their wild-type (Wt) littermates. Data are normalized per milligram of protein and expressed as means ± se. Inset: mitochondrial enzyme activity of CI, SDH, and GR after normalization by CS at 7 mo of age between male P301S mice and their wild-type male littermates (A). Starting at 7 mo, P301S mice had mitochondrial abnormalities compared to their wild-type littermates.