Original contributionInhibition of protein phosphatase 2A- and protein phosphatase 1-induced tau hyperphosphorylation and impairment of spatial memory retention in rats
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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2020, Progress in NeurobiologyCitation Excerpt :Dysregulation of the kinase/phosphatase system leads to the formation of hyperphosphorylated tau, a pathological form of tau that carries 3–4 times the normal number of phosphate groups (Iqbal et al., 2013). Inhibition of PP2A by okadaic acid and calyculin also results in the hyperphosphorylation of tau both in vitro by okadaic acid in metabolically active brain sections and SH-SY5Y cells and in vivo by calyculin (Sun et al., 2003; Gong et al., 2000; Zhang and Simpkins, 2010). Rats exposed to calyculin show a loss of spatial memory as measured by the Morris water maze test, manifested by performing a random search for the platform as opposed to a straight path to a pre-learned position (Sun et al., 2003).