Elsevier

Neuroscience

Volume 118, Issue 4, 6 June 2003, Pages 1175-1182
Neuroscience

Original contribution
Inhibition of protein phosphatase 2A- and protein phosphatase 1-induced tau hyperphosphorylation and impairment of spatial memory retention in rats

https://doi.org/10.1016/S0306-4522(02)00697-8Get rights and content

Abstract

Tau hyperphosphorylation leads to formation of paired helical filament/neurofibrillary tangles, the hallmark lesion seen in Alzheimer’s disease (AD) brain. An imbalanced regulation in protein kinases and protein phosphatases in the affected neurons is proposed to be a reasonable causative factor to the disease process. To verify the hypothesis, we have injected in the present study calyculin A, a potent and specific inhibitor of protein phosphatase (PP) 2A and PP1, into rat hippocampus bilaterally, thus reproduced an Alzheimer’s-like deficiency in dephosphorylation system. It was found that calyculin A-injected rats developed lesions in spatial memory retention in Morris water maze test. At mean time, tau was hyperphosphorylated at Ser396/Ser404 (PHF-1) and Ser-262/Ser-356 (12E8) sites determined both by immunohistochemistry and Western blot. It is implicated that (1) PP2A and PP1 participate in the in vivo regulation of tau phosphorylation, and down-regulation of the two phosphatases will result in tau hyperphosphorylation; (2) hyperphosphorylation of tau at PHF-1 and 12E8 sites might be crucial to affect spatial memory in AD.

Section snippets

Experimental procedures

All animal experiments were performed according to the “Policies on the Use of Animals and Humans in Neuroscience Research,” revised and approved by the Society for Neuroscience in 1995.

Materials

Antibodies to tau are listed in Table 1. Secondary antibodies for Western blot were from Amersham Pharmacia Biotech (Little Chalfort, Buckinghamshire, England). Detection kit (Histostain-SP) for immunohistochemistry was from ZEMED (South San Francisco, CA, USA). CA (Sigma, St Louis, MO, USA) was dissolved in 1% dimethylsulfoxide (DMSO) (V/V) with a stock concentration of 320 μM and stored at −20°C. For instant application, the stock solution of CA was then diluted in saline with a final

Injection

Forty-eight Sprague–Dawley rats, male, 3–5 months old, 250–350 g, were deeply anesthetized intraperitoneally with 5% chloral hydrate and placed in a stereotaxic instrument; 2 μl of CA or vehicle control (VC) consisted of normal saline and 0.05% DMSO was slowly injected into hippocampus with a 5-μl microsyringe using coordinates from the Paxinos atlas as follows (Paxinos et al., 1985): 4.8 mm anterior to posterior Bregma, 2.2 mm mid to lateral, 3.0 dorsal to ventral dura. The needle was left in

Immunohistochemistry

Rats were deeply anesthetized and transcardially perfused with 100 ml 0.01-M PBS, pH 7.4, first and then 400 ml 4% paraformaldehyde solution. The brain was dissected out and chopped into a 5×3×3-mm3 cube containing hippocampus. The cube was postfixed in the same 4% paraformaldehyde solution for 10 h before it was coronally sliced into 40-μm sections on a vibratome (LANCER, S100, TPI, Germany). Free-floating sections were blocked with 0.3% H2O2 in absolute methanol for 20 min and non-specific

Western blot

Rat hippocampi were quickly dissected out with cold homogenizing buffer containing 50-mM Tris–HCl, pH 7.0, 10-mM β-mercaptoethanol, 1.0-mM EDTA, 0.1-mM phenylmethylsufonyl fluoride, and 2.0 μg/ml each of aprotinin, leupeptin, and pepstatin A. Then they were homogenized in the same buffer at a ratio of 9.0 ml of buffer/1.0 g tissue with phosphatase inhibitor mixture containing 20-mM β-glycerophosphate, 2.0-mM Na3VO4, and 100-mM NaF, pH 7.0. The homogenates were spin at 15,000 r.p.m. for 3 min at

Step-down inhibitory avoidance task

The method was modified according to Netto and Izquierdo (1985) and Wilensky et al. (2000). Briefly, the experimental device is a 30-cm×30-cm×30-cm electronic avoidance-response chamber, made of Plexiglas on three sides and hard black plastic on the other. The chamber has a bottom of parallel 0.5-cm stainless steel bars spaced 1 cm apart. A rubber platform (5 cm high, 5 cm in diameter of its top surface) was randomly placed on the bottom of the chamber, providing rats a shelter from the

Morris water-maze test

The water-maze tests were conducted as described previously Bourtchuladze et al 1994, Guzowski and Mcgaugh 1997. Rats tested in the water maze were extensively handled (2 min every day for 7 days). Before each experiment (2 h), the rats were brought to the site to allow them to be acclimatized. The test subjects were kept in cages on outer-room shelves to eliminate directional olfactory and auditory cues. The temperature of the room and water was kept at 26±2 °C. The water in the pool is made

Effect of CA on step-down electronic inhibitory avoidance task of rats

To examine whether injection of CA would result in any behavioral dysfunction, we first detected how the rats acted in a one-trial step-down inhibitory avoidance task as a basal screening experiment (N=12). It was shown that the average response of CA-injected rats was similar to that of VC group (62.4±18.1 s versus 59.7±19.7 s; P=0.7). When daily response of each group was depicted into a learning curve, both CA- and VC-injected rats exhibit a remarkable learning process (Table 2, Fig. 1),

Discussion

In Alzheimer’s brain, tau can be isolated from different pools: (1) a cytosolic fraction; (2) soluble abnormally phosphorylated tau (P-tau); and (3) as a component of PHF (PHF-tau) (Kopke et al., 1993). A pool of soluble non-PHF but abnormally hyperphosphorylated tau suggests that the aberrant phosphorylation of tau precedes its polymerization into PHF. Thus, the cellular mechanisms by which tau becomes abnormally hyperphosphorylated and assembles into PHF will surely provide profound insights

Acknowledgements

We thank Drs. Khalid Iqbal and Inge Grundke-Iqbal from NYS Institute for Basic Research, Staten Island, New York, for their consistent scientific direction and material assistance. This work was supported in part by grants from the Natural Science Foundation of China (39925012, 39970808), Science and Technology Committee of China (G1999054007), National Educational Committee of China (2001-171).

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