An okadaic acid-induced model of tauopathy and cognitive deficiency

Abstract

Alzheimer's disease (AD) is a progressive neurodegenerative disease that causes cognitive and behavioral deterioration in the elderly. Neurofibrillary tangles (NFTs) are one of the pathological hallmarks of AD that has been shown to correlate positively with the severity of dementia in the neocortex of AD patients. In an attempt to characterize an in vivo AD tauopathy model, okadaic acid (OA), a protein phosphatase inhibitor, was microinfused into the right lateral dorsal hippocampus area of ovariectomized adult rat. Cognitive deficiency was seen in OA-treated rats without a change in motor function. Both silver staining and immunohistochemistry staining revealed that OA treatment induces NFTs-like conformational changes in both the cortex and hippocampus. Phosphorylated tau as well as cyclin-dependent kinase 5 (cdk5) and its coactivator, p25, were significantly increased in these regions of the brain. Oxidative stress was also increased with OA treatment as measured by protein carbonylation and lipid peroxidation. These data suggest that the unilateral microinfusion of OA into the dorsal hippocampus causes cognitive deficiency, NFTs-like pathological changes, and oxidative stress as seen in AD pathology via tau hyperphosphorylation caused by inhibition of protein phosphatases.

Fig. 1

MWM performance of rats infused with OA for 14 days. (A) The behavior test in the MWM started after 14 days of microinfusion of OA or vehicle into the dorsal hippocampus of the rats. The swimming path length to the escape platform was recorded to assess learning ability (acquisition test) and memory ability (retention test). After regular MWM, a visible MWM test (B) was performed and followed by rotarod tests (C). Control: 1% DMSO in artificial spinal–cerebral fluid, n=20. Treatment groups: OA (7 or 70 ng/day) was microinfused into unilateral dorsal hippocampal area for 14 days, n=10/group. Data were represented in mean±SEM. **p<0.01 between control and OA (70 ng/day) groups.

Fig. 2

Bielschowsky silver staining of the cortex in OA-treated rats. After the behavioral tests, half of rats received transcardiac perfusion with 4% formaldehyde in PBS, followed by immersion fixation of the removed brain. Then brain tissue was processed for paraffin sectioning, and 10-μm slides were prepared for silver staining. There was no positive staining found in either contralateral (A, B) or ipsilateral (C) cortex of control group. A few silver positive staining was found in the ipsilateral side of the cortex received low-dose OA (7 ng) infusion (F) but was not seen in the contralateral cortex (D, E). Much more silver positive staining was found in the both sides of cortex in high-dose OA group (G, H, I, K). The brain infusion cannula terminal was verified by H&E staining (J).

Fig. 3

Bielschowsky silver staining of the hippocampus in OA-treated rats. There was no positive silver staining found in the hippocampus of either control group (A, B, C) or low-dose OA (7 ng) group (D, E, F). A few silver positive staining was found in the dentate gyrus area of ipsilateral hippocampus from rats that received high-dose infusion of OA (70 ng) (I) but not in the contralateral side (G, H).

Fig. 4

Immunohistochemistry staining of the cortex in OA-treated rats. Fourteen days after microinfusion of OA into dorsal hippocampus unilaterally, rats were subjected to paraffin section preparation for immunohistochemistry staining and probed with antibody raised against phospho-tau (p-Thr²⁰⁵). There was no positive staining found in either side of the cortex in the control group (A, B, C). A few anti-p-Thr²⁰⁵ immunoreactivity positive staining was found on both side of the cortex in the rats that received low-dose OA infusion (D, E, F) and more positive staining was found in high-dose OA group (G, H, I).

Fig. 5

Immunohistochemistry staining of the hippocampus in OA-treated rats. Fourteen days after microinfusion of OA into dorsal hippocampus unilaterally, rats were subjected to paraffin section preparation for immunohistochemistry staining and probed with antibody raised against phospho-tau (p-Thr²⁰⁵). There was no positive staining found in either side of the hippocampus in the control group (A, B, C) or low-dose OA (7 ng) group (D, E, F). A few anti-p-Thr²⁰⁵ immunoreactivity in positive stained neurons were found in the CA1 area of the contralateral hippocampus of rats that received high-dose infusion of OA (70 ng) (G, H), and the ipsilateral hippocampus CA1 area was damaged (I).

Fig. 6

Tau phosphorylation in OA-treated rats. Fourteen days after microinfusion of OA into dorsal hippocampus unilaterally, rats were decapitated, and the brains were removed. The cortex and hippocampus were separated, homogenized in RIPA buffer, and centrifuged. The supernatants from different treatment groups were further subjected to Western blot for assessing the ratio of phospho-tau (T205) over total tau (T1) in both the hippocampus (A) and the cortex (B). Data are presented as mean±SEM for n=5. *p<0.05 and **p<0.01.

Fig. 7

GSK3β levels in OA-treated rats. Fourteen days after microinfusion of OA into dorsal hippocampus unilaterally, rats were decapitated, and the brains were removed. The cortex and hippocampus were separated, homogenized in RIPA buffer, and centrifuged. Supernatants from different treatment groups were further subjected to Western blot for assessing the ratio of p-GSK3β (Ser 9) over total GSK3β in both the hippocampus (A) and the cortex (B). Data are presented as mean±SEM for n=5. *p<0.05 and **p<0.01.

Fig. 8

Cdk5 and p25 levels in OA-treated rats. Fourteen days after microinfusion of OA into dorsal hippocampus unilaterally, rats were decapitated, and the brains were removed. The cortex and hippocampus were separated, homogenized in RIPA buffer, and centrifuged. Supernatants from different treatment groups were further subjected to Western blot for assessing the levels of cdk5 and p25 in both the hippocampus (A, C) and the cortex (B, D). Data were represented as mean±SEM for n=5. *p<0.05 and **p<0.01.

Fig. 9

MDA content in OA-treated rats. After behavioral tests, as described in Fig. 2, half of the rats were assessed for MDA concentrations. Cortex and hippocampus were separated and homogenized in specific buffer, and then the supernatants were collected for the lipid peroxidation assay as described in the Experimental procedures. Control: 1% DMSO in artificial spinal–cerebral fluid, n=4. Treatment groups: OA (7 or 70 ng/day) in vehicle, n=4/group. Data were represented in mean±SEM. *p<0.05 and **p<0.01.

Fig. 10

Protein carbonyl content in OA-treated rats. The cortex and hippocampus tissue from different treatment groups were homogenized, and the supernatants were collected for the protein carbonyl assay as described in the Experimental procedures. Control: 1% DMSO in artificial spinal–cerebral fluid, n=4. Treatment groups: OA (7 or 70 ng/day) in vehicle, n=4/group. Data were represented in mean±SEM. *p<0.05 and **p<0.01.