Selective changes of calcineurin (protein phosphatase 2B) activity in Alzheimer's disease cerebral cortex.

Neurofibrillary tangles, which contain abnormally hyperphosphorylated forms of tau protein, are one of the neuropathological hallmarks of Alzheimer's disease (AD). This altered phosphorylation state of tau protein may be due to increased kinase activity or/and decreased phosphatase activity. In the present study, we characterized human calcineurin phosphatase activity in postmortem superior frontal cortex and sensorimotor cortex and measured calcineurin phosphatase activity in samples from individuals with moderate to severe AD (n = 7) and age-matched controls (n = 5). Basal phosphatase activity was reduced by 25% (P < 0.05) in AD frontal cortex. Nickel-stimulated calcineurin activity was decreased by 52% (P < 0.05) and 30% (P < 0.05) in P2 and total cell homogenate, respectively, compared to age-matched controls. No differences in phosphatase activities were detected in the sensorimotor cortex. The decrease in nickel-stimulated calcineurin phosphatase activity in frontal lobe correlated with the neurofibrillary tangle pathology (total cell homogenate, r = -0.77, P < 0.05; P2 fraction, r = -0.76, P < 0.02), but not with diffuse or neuritic plaques. Despite the changes in calcineurin phosphatase activity in the superior frontal cortex, calcineurin protein levels determined by immunoblot were similar in control and AD cases. In addition, no changes in calcineurin regulatory proteins (cyclophilin A and FKBP12) levels were observed. These studies suggest that decrease of calcineurin activity may play a role in paired-helical filament formation and/or stabilization, and the decrease of activity was not accompanied by a decrease of calcineurin protein expression.

Effects of posttraining ethanol on an appetitive task.

The present study examined the effects of posttraining ethanol administration upon retention of an appetitive task using a variety of retention behaviors associated with the task. Male C57BL/6J mice were individually trained to find a cheese pellet placed in the corner of an open field. Five behavioral measures were used including locomotor activity counts, rearings, grooming episodes, approaches to the cheese pellet, and latency to consume the cheese pellet. Immediately after training, mice were injected intraperitoneally with saline or 2.0 g/kg of ethanol and then returned to their home cage in which four "intruder" mice were added for 2 h after training. On subsequent testing days (1, 6, 14, and 51 days posttraining), mice were returned to the original training environment and the five behaviors were measured. Both saline- and ethanol-treated mice habituated to the initially novel test environment at similar rates as indicated by decreased exploratory behavior (locomotor activity and rearings). In contrast, a divergence in the latency to consume the cheese pellet was observed: Saline-treated mice behaved as though the cheese was rewarding (decreased latency to eat the pellet), while the ethanol group behaved as though the cheese was aversive (increased latency to eat the pellet). Taken with previous studies, these results demonstrate that posttraining ethanol can have strikingly different effects on retention depending on the task, the measure of retention used, and the underlying neural structures involved.

Reduced high-affinity agonist binding at the M(1) muscarinic receptor in Alzheimer's disease brain: differential sensitivity to agonists and divalent cations.

M(1) muscarinic acetylcholine receptor (M(1)AchR)-G protein coupling, as measured by high-affinity agonist binding, was examined in membranes prepared from postmortem human temporal cortex (Brodmann area 38) from individuals with Alzheimer's disease (AD, n = 8) and age-matched controls (n = 6). Binding competitions between the M(1)AchR-selective antagonist [(3)H]pirenzepine ([(3)H]PZ) and muscarinic agonists carbachol, acetylcholine, oxotremorine, and oxotremorine M were conducted. In the presence of 1 mM MgCl(2), the inhibition of [(3)H]PZ binding by carbachol, acetylcholine, or oxotremorine M was best described by a two-affinity state model for control and AD cases, while oxotremorine binding affinity was best fit to a single-state model. Although both control and AD groups had similar K(D) values for the high- and low-affinity agonist binding sites, the proportion of M(1)AchRs exhibiting high affinity for carbachol and acetylcholine was reduced by 48 and 33%, respectively, in AD membranes relative to controls (P < 0.05). No changes in the binding of the oxotremorine M or oxotremorine were noted. The nonhydrolyzable guanine nucleotide GppNHp (100 microM) reduced the proportion of M(1)AchRs with high affinity for agonists in both control and AD membranes. Substitution of 1 mM MnCl(2) for MgCl(2) restored high-affinity carbachol binding at the M(1)AchR in AD membranes similar to that seen in controls. In the presence of 1 mM MnCl(2), agonist binding in controls did not differ from 1 mM MgCl(2). In the absence of cations (1 mM EDTA), no differences between control and AD M(1)AchR carbachol binding were observed. Thus, the loss of high-affinity agonist binding at the M(1)AchR in AD is dependent on the agonist and cation studied.

Reduction of calcineurin enzymatic activity in Alzheimer's disease: correlation with neuropathologic changes.

Neurofibrillary tangles (NFT), neuritic plaques, and dystrophic neurites are the classic neuropathologic hallmarks of Alzheimer's disease (AD), all of which contain to varying degrees abnormally and/or hyperphosphorylated forms of the microtubule-associated protein tau. Protein phosphatase 2B (calcineurin) dephosphorylates tau isolated from AD brains to control levels in vitro as well as regulates tau phosphorylation and function in vivo. It has been hypothesized that the changes in tau phosphorylation observed in AD may be due to increases in kinase activity and/or decreases in phosphatase activity. In order to investigate the latter possibility, we examined calcineurin enzyme activity using the substrate para-nitrophenyl-phosphate (pNPP) in postmortem brain samples from individuals with moderate to severe AD (n = 8) and age-matched controls (n = 7). The stimulation of calcineurin activity by manganese chloride (1 mM) was reduced by 60% (p < 0.01) in whole-cell homogenates prepared from AD temporal cortex (Brodmann area 38). On the other hand, in P2 membrane fractions, the stimulation of calcineurin activity by manganese chloride as well as nickel chloride (1 mM) was reduced by 37% (p < 0.05) and 79% (p < 0.01), respectively. The manganese-stimulated calcineurin activity in the temporal cortex inversely correlated with both the number of NFT (r = -0.60, p < 0.02) and neuritic/core plaques (r = -0.63, p < 0.02) in whole-cell homogenates, but only with NFT (r = -0.61, p < 0.02) in P2 membrane fractions. The nickel-stimulated calcineurin activity did not correlate with neuropathology measures in either whole-cell or P2 membrane fractions. In striate visual cortex (Brodmann area 17), an area relatively unaffected in AD, neither whole-cell nor P2 membrane calcineurin activity were significantly altered. To our knowledge, this is the first report of a reduction in calcineurin phosphatase activity in AD which correlates with the neuropathological features in a region-, subcellular fraction-, and divalent cation-specific manner.

Regional alterations in M1 muscarinic receptor-G protein coupling in Alzheimer's disease.

Previous studies examining the functional status of cortical muscarinic cholinergic M1 receptors have demonstrated an impairment in receptor-G protein coupling in Alzheimer's disease (AD) as measured by the inability of the receptor to form a high affinity agonist binding site. In order to investigate whether this alteration was a global phenomenon or a regional specific defect in signal transduction, we examined agonist binding at M1 receptors in three brain areas (superior frontal cortex, Brodmann areas 8 and 9; primary visual cortex, Brodmann area 17; and the dorsal striatum) within the same brain in controls and moderate to severe AD cases. Competition binding studies using the M1 antagonist 3H-pirenzepine (4 nM) in the presence of varying concentrations of the cholinergic agonist carbachol (50 nM to 1 mM) were performed in the presence and absence of GppNHp (100 microM), a non-hydrolyzable analog of GTP. In control membrane preparations, computer-assisted analysis of antagonist-agonist competition curves revealed that M1 receptor agonist binding fit a two site model with high and low affinity states in all three brain areas in the absence of GppNHp but only a single site in the presence of GppNHp. This is consistent with the ternary complex model of G protein-linked receptors. In contrast, curves obtained from both cortical regions from AD brains fit a single site model with low affinity in the presence or absence of GppNHp. On the other hand, agonist binding data obtained from the dorsal striatum of AD cases exhibited a two site fit, similar to that seen in controls.(ABSTRACT TRUNCATED AT 250 WORDS)