Subacute to chronic Alzheimer-like alterations after controlled cortical impact in human tau transgenic mice

Abstract

Repetitive traumatic brain injury (TBI) has been linked to late life development of chronic traumatic encephalopathy (CTE), a neurodegenerative disorder histopathologically characterized by perivascular tangles of hyperphosphorylated tau at the depth of sulci to later widespread neurofibrillary pathology. Although tau hyperphosphorylation and neurofibrillary-like pathology have been observed in the brain of transgenic mice overexpressing human tau with aggregation-prone mutation after TBI, they have not been consistently recapitulated in rodents expressing wild-type tau only. Here, we characterized Alzheimer-like alterations behaviorally, biochemically and immunohistochemically 6 weeks and 7 months after unilateral mild-to-moderate controlled cortical impact (CCI) in 5-7-month-old Tg/htau mice, which express all six isoforms of non-mutated human tau in a mouse tau null background. We detected hyperphosphorylation of tau at multiple sites in ipsilateral hippocampus 6 weeks but not 7 months after CCI. However, neuronal accumulation of AT8 positive phospho-tau was sustained in the chronic phase, in parallel to prolonged astrogliosis, and decreased neural and synaptic markers. The mice with CCI also exhibited cognitive and locomotor impairment. These results indicate subacute to chronic Alzheimer-like alterations after CCI in Tg/htau mice. This is the first known study providing insight into the role of CCI in Alzheimer-like brain alterations in young adult mice expressing only non-mutated human tau.

Figure 1

Study design. Tg/htau mice, 5–7 months of age, were subjected to controlled cortical impact (CCI) of the left parietal cortex. Neurobehavioral tasks, which included open field, elevated plus maze, novel object recognition, rota-rod and Morris water maze tests, were performed between 3 and 6 weeks after injury. One half number of mice were euthanized at 6 weeks and the remaining mice at 7 months after CCI for the assessment of subacute and chronic biochemical/immunohistological alterations of the brain, respectively. The insert is a schematic diagram showing the site of craniotomy and cortical impact.

Figure 2

CCI leads to impairment in motor coordination but not in spontaneous locomotor activity or anxiety in Tg/htau mice. (A–C) Elevated plus maze to test anxiety. (D–F) Open field test to assess spontaneous activity and anxiety-like behavior. (G) Accelerating rota-rod test to examine motor strength/coordination. Mice with CCI showed significant decrease in latency to fall off the rotating rod, as compared to sham control mice. Data are expressed as mean ±SD (n = 13–15 mice/group) and analyzed by unpaired Student t test (A–F) or repeated measures ANOVA followed by Bonferroni’s post hoc test (G). ***P < 0.001.

Figure 3

CCI impairs learning and memory in Tg/htau mice. (A,B) Novel object recognition to evaluate episodic memory. (C–E) Morris water maze to test learning and spatial reference memory. Mice after CCI showed significant impairment in episodic and spatial reference memories and reduced swimming speed, as compared to sham control mice. Data are expressed as mean ±SD (n = 13–15 mice/group) and analyzed by matched observation ANOVA followed by Bonferroni’s post hoc test (A), unpaired Student t test (B,C) or repeated measures ANOVA followed by Bonferroni’s post hoc test (D,E). *P < 0.05, ***P < 0.001.

Figure 4

CCI induces accumulation of hyperphosphorylated tau in the brain of Tg/htau mice. (A) Western blots and (B) quantification showing the level of tau phosphorylated at the indicated sites in the hippocampus 6 weeks after CCI. (C) Representative photomicrographs showing AT8 staining in the ipsilateral dentate gyrus (DG) and cerebral cortex 6 weeks after CCI. Numerous AT8-positive neuronal profiles were seen in the hilus and the ipsilateral cortex. (D) Western blots and (E) quantification showing the level of phosphorylated tau at indicated sites in the hippocampus 7 months after CCI. (F) Representative photomicrographs showing AT8 staining in DG and cerebral cortex 7 m after CCI. Data are expressed as mean ±SD (n = 5–7 mice/group) and analyzed by matched observation ANOVA followed by Bonferroni’s post hoc test. **P < 0.01 for phosphorylation sites in which interaction showed significant difference. Inserts show high magnification views correspondingly. Bar = 100 μm for all low magnification views and 20 μm for all inserts. Ips ipsilateral, Cont contralateral.

Figure 5

Alternative splicing of tau exon 10 is not dysregulated in CCI mouse brain on a subacute or chronic basis. (A) Western blots and (B) quantification showing the levels of 3R-tau, 4 R tau and total tau in the hippocampus 6 weeks after CCI. (C) Western blots and (D) quantification data showing the levels of 3R-tau, 4 R tau and total tau in the hippocampus 7 months after CCI. Data are expressed as mean ± SD (n = 5–7 mice/group) and analyzed by matched observation ANOVA followed by Bonferroni’s post hoc test. **P < 0.01. Ips ipsilateral, Cont contralateral.

Figure 6

The hippocampus shows decreased neuronal/synaptic markers after CCI in Tg/htau mice. (A) Western blots and (B) quantification showing levels of synapsin 1, synaptophysin, PSD-95 and NeuN in the hippocampus 6 weeks after CCI. (C) Western blots and (D) quantification data showing the levels of synapsin 1 and NeuN in the hippocampus 7 m after CCI. Data are expressed as mean ± SD (n = 5–7 mice/group) and analyzed by matched observation ANOVA followed by Bonferroni’s post hoc test. *P < 0.05, **P < 0.01. Ips ipsilateral, Cont contralateral.

Figure 7

CCI induces astrogliosis in the brain of Tg/htau mice. (A) Western blots and (B) quantification showing the level of GFAP in the hippocampus 6 w after CCI. (C) Immunostaining of mouse brain sections for GFAP 6 w after CCI. (D) Western blots and (E) quantification showing GFAP level in the hippocampus 7 m after CCI. (F) Immunostaining of mouse brain for GFAP 7 m after CCI. Data are expressed as mean ± SD (n = 5–7 mice/group) and analyzed by matched observation ANOVA followed by Bonferroni’s post hoc test. *P < 0.05, ***P < 0.001, ipsilateral v.s. contralateral; ^#P < 0.05, ^###P < 0.001 v.s. sham. Inserts show high magnification views correspondingly. Bar = 100 μm for all low magnification views and 20 μm for all inserts. Ips ipsilateral, Cont contralateral.