Sustained cholinesterase inhibition in AD patients receiving rivastigmine for 12 months.

OBJECTIVE: To study the long-term dual inhibitory effects of rivastigmine on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in patients with AD. METHODS: Eleven patients with mild AD received rivastigmine for 12 months. Cholinesterase (ChE) activities in the CSF and plasma were assessed colorimetrically. Immunoblot analysis was used to evaluate AChE isoforms. Neuropsychiatric tests were performed throughout the study. RESULTS: At 12 months, the mean dose of rivastigmine was 8.6 mg/d and specific activities of ChE in the CSF were lower than baseline values (by 36% for AChE and 45% for BuChE), correlating with parallel reductions in the plasma (27% for AChE and 33% for BuChE). The reduction of specific activities in the CSF, but not in the plasma, appeared to be dependent on the dose and duration of treatment. Scores of some of the neuropsychological tests associated with memory and attention were correlated with both plasma and CSF AChE and BuChE inhibition for up to 6 months. Immunoblot analysis revealed up-regulation of the "read-through" AChE isoform (AChE-R), whereas levels of the synaptic isoform were unchanged. CONCLUSIONS: Rivastigmine causes persistent inhibition of AChE and BuChE in CSF as well as plasma. The persistent CSF inhibition contrasts with earlier findings after long-term treatment by the reversible ChE inhibitor tacrine, which demonstrated increased AChE activity in the CSF but not in the blood. Rivastigmine's effects on the preferential up-regulation of the AChE-R isoform may have a favorable effect on disease stabilization.

The cerebrocortical areas in normal brain aging and in Alzheimer's disease: noticeable differences in the lipid peroxidation level and in antioxidant defense.

The markers of oxidative stress were measured in four cerebrocortical regions of Alzheimer's disease (AD) and age-matched control brains. In controls the levels of diene conjugates (DC) and lipid peroxides (LOOH) were significantly higher in the sensory postcentral and occipital primary cortex than in the temporal inferior or frontal inferior cortex. The antioxidant capacity (AOC) was highest in the temporal, and GSH in the frontal inferior cortex. The highest activity of superoxide dismutase (SOD) and catalase (CAT) was found in the occipital primary cortex. Compared with controls, significantly higher level of DC and LOOH and attenuated AOC were evident in AD temporal inferior cortex. In AD frontal inferior cortex moderate increase in LOOH was associated with positive correlation between SOD activity and counts of senile plaques. Our data suggest that in AD cerebral cortex, the oxidative stress is expressed in the reducing sequence: temporal inferior cortex > frontal inferior cortex > sensory postcentral cortex approximately = occipital primary cortex, corresponding to the histopathological spreading of AD from the associative to primary cortical areas.

Soluble interleukin-1 receptor type II levels are elevated in cerebrospinal fluid in Alzheimer's disease patients.

Evidence from epidemiological, clinical and experimental studies favour the hypothesis that inflammatory events are part of the neuropathology in Alzheimer's disease. Proinflammatory cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) have been found in activated microglia in the vicinity of amyloid plaques in Alzheimer's disease brain. In the present study, the levels of soluble IL-1 receptor type II (sIL-1R type II), IL-1 receptor antagonist (IL-1ra), IL-1beta, IL-6 and TNF-alpha were analyzed in cerebrospinal fluid (CSF) samples from Alzheimer's disease patients and control subjects. The levels of sIL-1R type II were significantly higher in CSF from Alzheimer's disease patients than in CSF samples from control subjects (38.5+/-8 pg/ml (mean+/-S.E.M.) vs. 7.9+/-4 pg/ml, p<0.05). Measurements of the proinflammatory cytokines IL-6 and TNF-alpha showed no significant difference between the two groups, and the levels of IL-1beta and IL-1ra in the present material were too low to permit detection. The increased levels of sIL-1R type II may reflect a compensatory mechanism to balance an increased release of IL-1 receptor agonists in the Alzheimer's disease brain.

Calcium ion transients in neutrophils from patients with sporadic Alzheimer's disease.

Abnormalities involving intracellular calcium homeostasis have been detected in Alzheimer's disease brain and fibroblasts as well as presenilin-1 mutation-bearing cells. In the present study we investigated inositol(1,4,5)trisphosphate-mediated calcium transients as well as calcium responses via mechanisms not related to surface receptors in Alzheimer's disease polymorphonuclear (PMN) granulocytes, using the tripeptide formyl-methionyl-leucyl-phenyl alanine (fMLP) and calcium ionophore ionomycin, respectively. fMLP elicited a biphasic response with an initial, fast increase in intracellular free calcium concentrations followed by a second, lower phase with no significant differences in either maximal response or time course between Alzheimer's disease granulocytes and controls. Similarly, the calcium signal elicited after ionomycin exposure was unchanged in Alzheimer's disease PMN. In conclusion, these results indicate that calcium mobilization from intracellular stores and via cross-membrane mechanisms is intact in Alzheimer's disease granulocytes.

Intracellular inositol (1,4,5)-trisphosphate receptor levels are preserved in Alzheimer's disease platelets.

An increasing number of signal transduction disturbances have been reported in Alzheimer's disease. These changes are not restricted to histopathologically changed brain areas but are seen also in peripheral tissues. One of the most severe disturbances is a loss of calcium-mobilizing intracellular inositol(1,4,5)-trisphosphate receptors in Alzheimer cerebellar and cortical tissues. In the present study, the binding of [3H]inositol(1,4,5)trisphosphate ([3H]Ins(1,4,5)P3) to the calcium-mobilizing inositol(1,4,5)trisphosphate receptors in platelet membranes from eight Alzheimer's disease patients and eight control subjects were investigated to determine its possible role as a biological marker in Alzheimer's disease. It was found that there were no significant difference in [3H]Ins(1,4,5)P3 binding with respect to the number of sites measured at different protein concentrations or to the sensitivity of the binding to inhibition by nonradioactive Ins(1,4,5)P3 between Alzheimer disease platelets and controls. It is concluded that inositol(1,4,5)trisphosphate receptor levels are preserved in platelets from patients with Alzheimer's disease.

Decreased beta-adrenoceptor-stimulated adenylyl cyclase activity in lymphocytes from Alzheimer's disease patients.

Previous studies have shown that in Alzheimer's disease post-mortem brain there are disruptions of both beta1-adrenoceptor-G-protein coupling and G-protein stimulation of adenylyl cyclase activity. Decreased beta-adrenoceptor stimulated adenylyl cyclase activity has also been shown in Alzheimer's disease primary skin fibroblasts. In the present study, we determined the regulation of adenylyl cyclase in Alzheimer's disease patients using an easily accessible tissue source, namely peripheral blood lymphocytes. beta-Adrenoceptor- and forskolin-stimulated adenylyl cyclase activities were investigated in lymphocytes from 12 Alzheimer's disease and 12 carefully matched and selected control subjects. No significant differences were found in basal or forskolin-stimulated enzyme activities between Alzheimer's disease and control lymphocytes. In contrast, isoprenaline-stimulated adenylyl cyclase activities were significantly lower in the Alzheimer's disease groups, as compared to controls. These results indicate that there is a widespread disruption of beta-adrenoceptor-G-protein-enzyme coupling in different tissues from Alzheimer's disease patients, and that adenylyl cyclase disturbances previously reported in Alzheimer's disease brain do not occur as a consequence of disease pathology or of terminal illness.

Receptor-effector coupling dysfunctions in Alzheimer's disease.

There is now good evidence that in the AD brain, a number of neurotransmitter effector systems are defective. Such abnormalities include defective G, protein and protein kinase C function as well as a drastically reduced level of receptors for the second messenger Ins(1,4,5) P3. Such changes are probably not restricted to the late stages of the disease, and are found in regions of the brain that show little histopathological abnormality, such as the cerebellum. Whether these changes precede or are secondary to primary histopathological changes such as beta-amyloid deposition is not as yet clear. What is clear, however, is that such signal transduction abnormalities are likely to negate therapeutic benefits in clinical strategies based upon the tenet of neurotransmitter replacement.

Decreased inositol (1,4,5)-trisphosphate receptor levels in Alzheimer's disease cerebral cortex: selectivity of changes and possible correlation to pathological severity.

We used immunoblotting and radioligand binding techniques to compare levels of the calcium-mobilizing receptor for the phosphoinositide hydrolysis-derived intracellular second messenger inositol (1,4,5)-trisphosphate (IP3) in post mortem samples from the temporal, frontal and parietal cortices of eight Alzheimer's disease (AD) and eight matched control cases. Immunoblotting with an antibody directed against the C-terminal end of the rat type I IP3-receptor showed that IP3-receptor protein levels were significantly reduced in the temporal (to 59 +/- 6% of controls, P = 0.0002) and frontal (to 62 +/- 10% of controls, P = 0.04), but not in the parietal cortices (to 63 +/- 13% of controls, P = 0.1) of the AD cases, compared to controls. The number of [3H]IP3 radioligand binding sites was significantly decreased in the temporal cortex, but not frontal and parietal cortices, of the AD brains. The decreased levels of both immunoreactive IP3-receptor protein and [3H]IP3 binding in the temporal cortex correlated with a semi-quantitative score for the severity of AD neuropathology. No significant changes were seen in the levels of glial fibrillary acidic protein, synaptophysin or phosphate-activated glutaminase, as markers for astrocytes, neuronal vesicles and mitochondria, respectively. It is concluded that in affected AD brain regions, the IP3-receptor may represent a sensitive target for proteolysis, possibly mediated by activation of the Ca(2+)-activated neutral protease calpain. These degenerative changes may in part be responsible for the disruption of Ca2+ homeostasis in AD-sensitive neurons.

Disturbances in signal transduction mechanisms in Alzheimer's disease.

Many of the treatments directed towards alleviation of symptoms in Alzheimer's disease assume that target receptor systems are functionally intact. However, there is now considerable evidence that this is not the case. In human post-mortem brain tissue samples, the function of the GTP-binding protein Gs in regulating adenylyl cyclase is severely disabled, whereas that of Gi is intact. This difference in the function of the two G-protein types is also found in G-protein regulation of high- and low-affinity receptor recognition site populations. Measurement of G-protein densities using selective antibodies has indicated that the dysfunction in Gs-stimulation of cAMP production correlates with the ratio of the large to small molecular weight isoforms of the Gs alpha subunit. With respect to intracellular second messenger effects, there is a dramatic decrease in the density of brain receptor recognition sites for Ins(1,4,5)P3 that is not accompanied by a corresponding change in the Ins(1,3,4,5)P4 recognition site density. Protein kinase C function is also altered in Alzheimer's disease, a finding that may be of importance for the control of beta-amyloid production. These studies indicate that signal transduction processes are severely compromised in Alzheimer's disease. Some of these disturbances are also seen in cultured fibroblasts from Alzheimer's disease patients, indicating that they are neither restricted to areas of histopathological change, nor non-specific changes found late in the course of the disease. Cellular models to investigate the relation between amyloid production and deficits in signal transduction are also discussed.

Diminished [3H]inositol(1,4,5)P3 but not [3H]inositol(1,3,4,5)P4 binding in Alzheimer's disease brain.

Levels of the calcium mobilising receptors for the phosphoinositide hydrolysis derived second messengers, inositol(1,4,5)trisphosphate [Ins(1,4,5)P3] and inositol(1,3,4,5) tetrakis-phosphate [Ins(1,3,4,5)P4] were compared in the cerebellum, superior temporal and superior frontal cortex of a series of Alzheimer's disease and matched control cases. Membrane [3H]Ins(1,4,5)P3 radioligand binding experiments performed under steady state conditions revealed that the number of Ins(1,4,5)P3 recognition sites was significantly decreased in all three brain regions of the Alzheimer's disease cases, compared to controls. In contrast, [3H]Ins(1,3,4,5)P4 binding levels, as assessed in competition analyses, were not significantly different between the groups in any brain region. Moreover, the Hill coefficients for inhibition of [3H]Ins(1,3,4,5)P4 binding by non-radioactive Ins(1,3,4,5)P4 were less than unity in both the control and Alzheimer's disease brains, suggesting that the heterogeneity of these binding sites are also maintained in the disease. It is concluded that disruptions of the phosphoinositide hydrolysis pathway in Alzheimer's disease brain are associated with a selective loss of calcium mobilising Ins(1,4,5)P3, but not Ins(1,3,4,5)P4 receptor sites. These alterations may contribute to an altered calcium homeostasis in Alzheimer's disease, as well as providing one reason for the lack of success of cholinergic replacement therapies aimed at enhancing muscarinic receptor-mediated phosphatidylinositol hydrolysis.

Preservation of kappa 1 opioid receptor recognition site density and regulation by G-proteins in the temporal cortex of patients with Alzheimer's disease.

The pharmacological properties of the kappa 1 opioid receptor were investigated in human post-mortem temporal cortical membranes from control and Alzheimer's disease brains, using the kappa 1-selective radioligand [3H]U69593. [3H]U69593 bound to a single high affinity site population with no significant difference between control (Bmax 31 +/- 4.14 fmol/mg protein, KD 1.01 +/- 0.26 nM) and Alzheimer's disease brains (Bmax 37 +/- 4.63 fmol/mg protein, KD 0.86 +/- 0.08 nM). Competition studies with dynorphin B and alpha-neoendorphin gave flat inhibition curves with Hill coefficients of 0.31 +/- 0.04 and 0.49 +/- 0.09 in the control brains and 0.38 +/- 0.05 and 0.48 +/- 0.08 in the Alzheimer's disease brains, respectively. The pI50 values for dynorphin B and alpha-neoendorphin were 8.73 +/- 0.17 and 8.48 +/- 0.09, respectively, in the control brains and 9.30 +/- 0.22 and 8.70 +/- 0.15 in the Alzheimer's disease brains. The guanine nucleotide analogue Gpp(NH)p inhibited binding by ca. 70% in both the control and Alzheimer's disease brains, the residual binding being sensitive to NaCl in both cases. These results indicate that the pharmacological properties and the functional integrity of G-protein coupling of the kappa 1 receptor recognition site are preserved in Alzheimer's disease temporal cortex.

Characteristics of [3H]inositol(1,3,4,5)tetrakisphosphate recognition sites in human cerebellar membranes.

The characteristics of specific [3H]Ins(1,3,4,5)P4 binding sites in human cerebellar membranes were determined in this study. Binding rapidly reached steady state, possessed a pH optimum of 4.5-5.1 and was greater in the absence of BSA than in its presence. Heparin inhibited both specific and pseudospecific binding of the ligand, whereas only the specific binding was inhibited by non-radioactive Ins(1,3,4,5)P4. Calcium at a concentration of 1 mM, reduced binding by 27%. Competition studies with other inositol phosphates showed specificity for Ins(1,3,4,5)P4 with a pI50 value of 6.87 and a Hill coefficient of 0.27, indicating two sites. Ins(1,2,5,6)P4, Ins(1,3,4,6)P5, Ins(3,4,5,6)P4 displaced binding with IC50 values ranging from 0.1-1 microM, Ins(1,2,5,6)P4 and Ins(1,3,4,5,6)P5 being the most potent. Ins(1,4)P2 and Ins(1,5,6)P3 had lesser effects on binding. Rosenthal analysis of [3H]Ins(1,3,4,5)P4 saturation binding data at low ligand concentrations gave a KD of 27 nM and a Bmax of 33 pmol/mg protein. It is concluded that [3H]Ins(1,3,4,5)P4 binding sites in human cerebellar membranes have similar characteristics to these sites reported in the literature in animal cerebellar tissue, but are in greater abundance.

Characterization of [3H]inositol 1,4,5-trisphosphate binding sites in human temporal cortical and cerebellar membranes.

The characteristics of [3H]Ins(1,4,5)P3 binding to human temporal cortical and cerebellar membranes have been determined and compared with the binding to calf cerebellar membranes. Association and dissociation of ligand was very rapid, k1 and k-1 values of the order of 7 x 10(7) M-1 min-1 and 0.2 min-1, respectively. KD values were 2.7 and 3.5 nM for temporal cortex and cerebellum, respectively. The corresponding Bmax values were 165 and 482 fmol/mg protein. Binding was influenced in a biphasic manner by calcium. The temporal cortical binding was inhibited by Ins(1,4,5)P3 and analogues with the following IC50 values (nM): Ins(1,4,5)P3 9.5 and 6.2 (two different salts from different sources), Ins(2,4,5)P3 42, Ins(1,3,4,5)P4 670, Ins(1,2,5,6)P4 2620, Ins(3,4,5,6)P4 4300, Ins(1,3,4,5,6)P5 5490, InsP(6)5280, Ins(4,5)P2 2600, Ins(1)P 3300, with the IC50 values for Ins(1,5,6)P3, Ins(1,4)P2 and Ins(4)P being > 25 microM. The IC50 value for heparin was 2.1 micrograms/ml. A similar pattern was seen in the cerebellum. In both tissues, the Hill slopes were near unity for all compounds except Ins(3,4,5,6)P4, where the slope was 0.4. The calf cerebellum had a similar ligand specificity (although the potency was generally lower) when values were expressed relative to that of Ins(1,4,5)P3, with the possible exception of Ins(1,3,4,5)P4, which had a greater relative potency. These data would suggest that in the human temporal cortex and cerebellum, [3H]Ins(1,4,5)P3 binding sites are expressed in different densities, but have similar properties. There may, however, be species differences in the [3H]Ins(1,4,5)P3 recognition site.

Neurotransmitter-mediated inhibition of post-mortem human brain adenylyl cyclase.

The effects of a range of neurotransmitter agonists showing selectivity for receptor types inhibitorily coupled to adenylyl cyclase were compared in membrane preparations of hippocampus, frontal cortex and caudate nucleus/striatum from previously frozen post-mortem human and rat brain. Agonists were tested against basal and forskolin stimulated activities, forskolin being a potent activator of the catalytic sub-unit of the enzyme. Of those agonists tested, only somatostatin (100 microM) and neuropeptide Y (10 microM) gave consistent inhibitions of basal and forskolin stimulated enzyme activities in all three regions of both human and rat brain. Somatostatin-mediated inhibition of human brain adenylyl cyclase was reduced in the absence of GTP and in the presence of the guanine nucleotide partial agonist, guanosine 5'-O-thiodiposphate, consistent with a G-protein-linked receptor. No such GTP-dependence was found for the neuropeptide Y-mediated adenylyl cyclase inhibition. GTP-dependent somatostatin mediated inhibitions of human brain adenylyl cyclase activity were of highest magnitude in the thalamus, intermediate magnitude in the hippocampus and caudate nucleus and lowest magnitude in the frontal cortex. It is concluded that of a range of neurotransmitter receptor agonists tested, only somatostatin gives robust, GTP-dependent responses that are reproducible enough to be used with post-mortem tissue for the comparison of receptor function in human brain disorders.

Regional distribution of somatostatin receptor binding and modulation of adenylyl cyclase activity in Alzheimer's disease brain.

We have previously reported a reduction in the inhibitory effect of somatostatin on adenylyl cyclase activity in the superior temporal cortex of a group of Alzheimer's disease cases, compared to a group of matched controls. In the present study, the levels of high affinity 125I-Tyr11-somatostatin-14 binding, its modulation by guanine nucleotides and the effects of somatostatin on adenylyl cyclase activity have been measured in preparations of frontal cortex, hippocampus, caudate nucleus and cerebellum from the same patient and control groups. A significant reduction in 125I-Tyr11-somatostatin-14 binding was observed in the frontal cortex, but not other regions, of the Alzheimer's disease group, compared with control values. The profiles of inhibition of specific 125I-Tyr11-somatostatin-14 binding by Gpp(NH)p were similar in all regions in both groups. No significant differences in basal, forskolin-stimulated, or somatostatin and neuropeptide Y inhibitions of adenylyl cyclase activity were found between the two groups. The pattern of change of somatostatin binding in the Alzheimer's disease cases observed in the present study differs from the reported pattern of loss of somatostatin neurons and may be secondary to the degeneration of somatostatin receptor-bearing cholinergic afferents arising from the nucleus basalis. The results of this study indicate that impaired somatostatin modulation of adenylyl cyclase is not a global phenomenon in Alzheimer's disease brain and also that there are no major disruptions of somatostatin receptor-G-protein coupling or of adenylyl cyclase catalytic activity in this disorder.

Characterization and regional distribution of adenylyl cyclase activity from human brain.

The characteristics of the human brain adenylyl cyclase complex were studied in membranes from post-mortem frontal cortex. Basal, guanine nucleotide, sodium fluoride and forskolin stimulated activities were highly magnesium-dependent. Sodium fluoride and guanine nucleotides gave bi-phasic responses, with both stimulation and inhibition of enzyme activity, the latter being more pronounced at lower magnesium concentrations. Enzyme activity was stimulated to a similar extent by GTP and its non-hydrolysable analogue Gpp(NH)p, suggesting a low GTPase activity in human post-mortem brain preparations. Guanine nucleotide stimulated enzyme activity was potently antagonized by guanosine 5?-O-(thiodiphosphate). Sodium fluoride stimulated activity was enhanced by aluminium chloride. In contrast to the effects seen with guanine nucleotides, the inhibition of sodium fluoride/aluminium chloride stimulated activity by guanosine 5?-O-(thiodiphosphate) was dependent upon pre-incubation of membranes with a neurotransmitter agonist. Basal, guanine nucleotide and sodium fluoride/aluminium chloride stimulated activities showed a marked regional distribution. Stimulated activities were highest in frontal and parietal cortex, intermediate in the nucleus caudatus and cerebellar cortex and lowest in occipital cortex, putamen, globus pallidus and ventral hippocampus. It is concluded that the regulation of human post-mortem brain adenylyl cyclase by guanine nucleotides is similar to that reported for studies on experimental animals.

Effect of monovalent ions upon G proteins coupling muscarinic receptors to phosphoinositide hydrolysis in the rat cerebral cortex.

It has been suggested that K+, Li+ and Fl- affect the function of G proteins coupled to signal transducing enzymes. Lithium, at concentrations which were found to reduce forskolin-stimulated adenylate cyclase activity, was without effect on either membrane [3H]phosphatidylinositol-4,5-bisphosphate ([3H]PIP2) hydrolysis measured in the absence or presence of 5'-guanylyl-imidodiphosphate (Gpp(NH)p), or (at greater than or equal to 2.3 mM Li+) upon the stimulation of rat cerebral cortical inositol phospholipid breakdown by either carbachol, noradrenaline or NaF measured at either 6 or 18 mM K+. The increase in assay [K+] greatly enhanced the inositol phospholipid response to carbachol but not to NaF. The inhibitory effect of carbachol upon forskolin-stimulated adenylate cyclase was not affected by raising the [K+] from 6 to 18 mM. At 6 mM K+ (both in the absence and presence of 15 microM AlCl3), the effects of carbachol and NaF upon inositol phospholipid breakdown were essentially additive, whereas at 18 mM K+, the breakdown response to carbachol (antagonised by pirenzepine with a pA2 value of 7.6) was similar in the absence and presence of NaF. It is concluded that in the rat cerebral cortex: (a) Li+ does not affect the function of either the phosphoinositide-specific phospholipase C enzyme itself or the Gp coupled to this enzyme; (b) the difference between the additivity between NaF and carbachol seen at different assay [K+] may reflect the K(+)-dependent changes in the tetrodotoxin-resistant and tetrodotoxin-sensitive pathways of carbachol stimulation of inositol phospholipid breakdown reported by Gurwitz and Sokolovsky (1987, Biochemistry 26, 633); and (c) the effect of K+ on muscarinic receptor-coupled inositol phospholipid breakdown is not found for muscarinic receptors inhibitorily coupled to adenylate cyclase. Evidence is also presented to suggest that NaF affects the dephosphorylation of the formed [3H]inositol polyphosphates.