Ello Karelson

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 Alzheimers 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 ≃ occipital primary cortex, corresponding to the histopathological spreading of AD from the associative to primary cortical areas.

The Swedish APP670/671 Alzheimer’s Disease Mutation: The First Evidence for Strikingly Increased Oxidative Injury in the Temporal Inferior Cortex

To evaluate the level of oxidative stress (OS) in familial Alzheimer’s disease (FAD), we analysed four cerebrocortical areas from patients with Swedish FAD bearing the APP670/671 mutation. The temporal inferior cortex (TIC) from Swedish FAD patients revealed a striking 2- to 3-fold increase in diene conjugates, lipid peroxides and protein carbonyls, compared to sporadic Alzheimer’s disease (AD). Compared with TIC from sporadic AD patients, the mutation carriers showed a markedly decreased activity of catalase (CAT) in the same area, and the same trend was found for another antioxidant enzyme, superoxide dismutase. These results are consistent with the deep oxidative injury of TIC in Swedish FAD. In the frontal inferior cortex (FIC), sensory postcentral cortex (SPCC) and occipital primary cortex (OPC) from Swedish FAD, the parameters of oxidative injury tended to be higher than in sporadic AD. Only the increase in the levels of lipid hydroperoxides in SPCC and of protein carbonyls in OPC was significant. Compared to sporadic AD, Swedish FAD showed a significant increase in GSSG levels and the GSSG/2GSH ratio in the FIC, SPCC and OPC. A significantly decreased activity of CAT was detectable for the SPCC and OPC in Swedish FAD. Increased OS might play a crucial role in the rapid progression of Swedish FAD from the associative temporal cortex to the primary cerebrocortical areas.

Possible Signaling by Glutathione and Its Novel Analogue through Potent Stimulation of Frontocortical G Proteins in Normal Aging and in Alzheimer's Disease

Abstract: In the frontal cortex (FC) of the normally aging human brain, glutathione (GSH) and its novel analogue, UPF1, stimulate G proteins more than in Alzheimers disease (AD) FC. In normal aging and in AD, UPF1 is a more efficient stimulator of G proteins than GSH. In normal FC, both GSH and UPF1 stimulate G proteins, which mediate inhibitory signals to the cAMP system; while in AD, only UPF1 exhibits the same action. Stimulation of G proteins and coupled signaling by GSH antioxidant analogues, as potential signaling molecules, may ameliorate the oxidative impairments of neuronal signaling in AD.

Differential regulation of adenylate cyclase activity in rat ventral and dorsal hippocampus by rat galanin

Rat galanin inhibits basal as well as forskolin-stimulated adenylate cyclase activity in rat ventral and dorsal hippocampus. The inhibition of adenylate cyclase activity, both basal and forskolin-stimulated, is characterised by IC50 values being 250-fold lower in ventral hippocampus (IC50 = 1.1 nM) compared to the dorsal hippocampus (IC50 = 270 nM). The maximal inhibition of basal and forskolin-stimulated adenylate cyclase activity in both ventral and dorsal hippocampus in the presence of 10 microM rat galanin is 34-45%. The analysis of the binding data obtained with 125I-labelled Tyr26-porcine galanin as a tracer reveals similar binding constants for rat galanin in both ventral and dorsal hippocampus with 4.8-fold higher concentration of galanin receptors in the ventral hippocampus. Putative galanin receptor subtype differences between the ventral and dorsal hippocampus have been noted by Hedlund et al. (Eur. J. Pharmacol., 224 (1992) 203-205). This study yields further confirmation for the existence of different galanin receptor subtypes or for differential coupling of galanin receptors to the adenylate cyclase in the dorsal versus ventral hippohampus.

Regulation of the frontocortical sodium pump by Na+ in Alzheimer's disease: difference from the age-matched control but similarity to the rat model

The Na+ and K+ dependence of the frontocortical Na,K‐ATPase in Alzheimers disease (AD) was compared with that in human control (Co) and rat AD model. In AD, the relationship between the Na/K ratio and the Na,K‐ATPase activity showed noticeable left‐shift with three‐fold increase in the enzyme affinity for Na+ (K 0.5=10 and 30 mM in AD and Co, respectively). The Na+ dependence of the enzyme in AD showed two different Hill coefficients (n H), 1.1 and 0.3, whereas the Co value of n H was higher (1.4). The rat AD model generated by ibotenic acid revealed a Na+ dependence similar to AD. The K+ dependence of the Na,K‐ATPase showed no significant difference in AD and Co. Compared with Co, AD produced a shift in the break of the Na,K‐ATPase Arrhenius plot, suggesting remarkable alterations in the enzyme lipid environment. Our findings support the hypothesis that dysfunction of the Na,K‐ATPase in AD is provoked by altered Na+ dependence of the enzyme. An impairment of the pump functionality might serve as an early mechanism of AD that should be interrupted by selective pharmacological agents.

Regulation of GTPase and adenylate cyclase activity by amyloid β-peptide and its fragments in rat brain tissue

Modulation of GTPase and adenylate cyclase (ATP pyrophosphate-lyase, EC 4.6.1.1) activity by Alzheimers disease related amyloid beta-peptide, A beta (1-42), and its shorter fragments, A beta (12-28), A beta (25-35), were studied in isolated membranes from rat ventral hippocampus and frontal cortex. In both tissues, the activity of GTPase and adenylate cyclase was upregulated by A beta (25-35), whereas A beta (12-28) did not have any significant effect on the GTPase activity and only weakly influenced adenylate cyclase. A beta (1-42), similar to A beta (25-35), stimulated the GTPase activity in both tissues and adenylate cyclase activity in ventral hippocampal membranes. Surprisingly, A beta (1-42) did not have a significant effect on adenylate cyclase activity in the cortical membranes. At high concentrations of A beta (25-35) and A beta (1-42), decreased or no activation of adenylate cyclase was observed. The activation of GTPase at high concentrations of A beta (25-35) was pertussis toxin sensitive, suggesting that this effect is mediated by Gi/G(o) proteins. Addition of glutathione and N-acetyl-L-cysteine, two well-known antioxidants, at 1.5 and 0.5 mM, respectively, decreased A beta (25-35) stimulated adenylate cyclase activity in both tissues. Lys-A beta (16-20), a hexapeptide shown previously to bind to the same sequence in A beta-peptide, and prevent fibril formation, decreased stimulation of adenylate cyclase activity by A beta (25-35), however, NMR diffusion measurements with the two peptides showed that this effect was not due to interactions between the two and that A beta (25-35) was active in a monomeric form. Our data strongly suggest that A beta and its fragments may affect G-protein coupled signal transduction systems, although the mechanism of this interaction is not fully understood.

Stimulation of G-proteins in human control and Alzheimer's disease brain by FAD mutants of APP714–723: Implication of oxidative mechanisms

We report the effects of amyloid precursor protein (APP) fragment 714–723 (APP714–723; peptide P1) and its V717F and V717G mutants (peptides P2 and P3, respectively) on G‐protein activity ([35S]GTPγS binding) in membranes from postmortem human control and Alzheimers disease (AD) brains. The peptides P1, P2, and P3 revealed a significant stimulatory effect on [35S]GTPγS binding in control temporal cortex. The most potent stimulator, P3, at 10 μM concentration enhanced [35S]GTPγS binding by 500%. The effect was threefold stronger than that for wild‐type P1 and twofold stronger than that for P2. In sporadic AD, the stimulatory effect of P1, P2, and P3 on G‐proteins was reduced significantly whereas in Swedish familial AD (SFAD), only P1 elicited marked stimulation (at 10 μM by 50%). In control sensory postcentral cortex, the stimulation of G‐proteins by P3 was 1.5‐fold lower than that in control temporal cortex, whereas in AD and SFAD the effect showed no remarkable regional difference. Treatment of membranes with H2O2 produced 1.5‐fold higher stimulation in [35S]GTPγS binding to temporal cortex than that in binding to sensory postcentral cortex. In AD and SFAD, the stimulation by H2O2 revealed no significant regional difference. Glutathione, desferrioxamine (DFO), and 17β‐estradiol markedly decreased the strong stimulatory effect by P3 on [35S]GTPγS binding to control temporal cortex, with the protective effect by DFO being most potent. The Gαo‐protein levels were not changed in AD or SFAD brain membranes as compared to levels in control membranes. We suggest that strong G‐protein stimulation by P3 in the human brain implies the specific (per)oxidation mechanism that might be affected by regional content of peroxidizing substrates and antioxidants.

Mitochondrial oxidative stress index, activity of redox-sensitive aconitase and effects of endogenous anti- and pro-oxidants on its activity in control, Alzheimer's disease and Swedish Familial Alzheimer's disease brain

Efficient function of the mitochondrial respiratory chain and the citric acid cycle (CAC) enzymes is required for the maintenance of human brain function. A conception of oxidative stress (OxS) was recently advanced as a disruption of redox signalling and control. Mitochondrial OxS (MOxS) is implicated in the development of Alzheimers disease (AD). Thus, both pro- and anti-oxidants of the human body and MOxS target primarily the redox-regulated CAC enzymes, like mitochondrial aconitase (MAc). We investigated the specific activity of the MAc and MOxS index (MOSI) in an age-matched control (Co), AD and Swedish Familial AD (SFAD) post-mortem autopsies collected from frontal cortex (FC) and occipital primary cortex (OC) regions of the brain. We also examined whether the mitochondrial neuroprotective signalling molecules glutathione, melatonin and 17-β-estradiol (17βE) and mitochondrially active pro-oxidant neurotoxic amyloid-β peptide can modulate the activity of the MAc isolated from FC and OC regions similarly or differently in the case of Co, AD and SFAD. The activity of redox-sensitive MAc may directly depend on the mitochondrial oxidant/antioxidant balance in age-matched Co, AD and SFAD brain regions.

17β-Oestradiol Stimulation of G-Proteins in Aged and Alzheimer’s Human Brain: Comparison with Phytoestrogens

The neuroprotective action of oestrogens and oestrogen‐like compounds is in the focus of basic and clinical research. Although such action has been shown to be associated with neuronal plasma membranes, the implication of G‐proteins remains to be elucidated. This study revealed that micromolar concentrations (μm) of 17β‐oestradiol and phytoestrogens, genistein and daidzein, significantly (P < 0.05) stimulate G‐proteins ([35S]GTPγS binding) in the post‐mortem hippocampal membranes of age‐matched control women with the respective maximum effects of 28, 20 and 15% at 10 μm. In the frontocortical membranes, the stimulation of G‐proteins did not differ significantly from that in hippocampal membranes. Although in the hippocampus and frontal cortex of the Alzheimer’s disease (AD) womens brain, 10 μm 17β‐oestradiol produced significantly (P < 0.05) lower stimulation of G‐proteins than in the control regions, stimulation by phytoestrogens revealed no remarkable decline. 17β‐Oestradiol, genistein and daidzein revealed a selective effect on various G‐proteins (Gαs, Gαo, Gαi1 or Gα11 plus Gβ1γ2) expressed in Sf9 cells. At a concentration of 10 μm, 17β‐oestradiol suppressed the H2O2 and homocysteine stimulated G‐proteins in the frontocortical membranes of control women to a greater extent than phytoestrogens. In AD, the suppressing effect of each compound was lower than in the controls. In the cell‐free systems, micromolar concentrations of phytoestrogens scavenged OH• and the 2.2‐diphenyl‐1‐picrylhydrazyl free radical (DPPH•) more than 17β‐oestradiol did. In the frontocortical membranes of control women, the 20 μm 17β‐oestradiol stimulated adenylate cyclase with 20% maximal effect, whereas, in AD, the effect was insignificant. Genistein did not stimulate enzyme either in control or AD frontocortical membranes. Our data confirm that the agents stimulate G‐proteins in control and AD womens brains, although 17β‐oestradiol and phytoestrogens have similarities and differences in this respect. We suggest that, besides the ER‐dependent one, the ER‐independent antioxidant mechanism is responsible for the oestrogen stimulation of G‐proteins in the brain membranes. Both of these mechanisms could be involved in the neuroprotective signalling of oestrogens that contributes to their preventive/therapeutic action against postmenopausal neurological disorders.

Characterisation of a new chimeric ligand for galanin receptors: galanin(1–13)-[d-Trp32]-neuropeptide Y(25–36)amide

In this work, we studied a novel chimeric peptide, M242, galanin(1-13)-[D-Trp(32)]-neuropeptide Y(25-36)amide, and examined its properties in comparison with its parent peptide, M32, galanin(1-13)-neuropeptide Y(25-36)amide, a previously known high-affinity ligand for galanin receptors, and galanin itself. Binding assays performed in Bowes cells known to express human galanin receptor type 1 (hGalR1) and in Chinese hamster ovary cells overexpressing human galanin receptor type 2 (hGalR2) revealed that all three ligands had comparable affinities: at hGalR1<1 nM and at hGalR2<10 nM. However, in rat hippocampal membranes M242 had a 24-fold lower affinity than galanin (9.4 vs. 0.4 nM) and 134-fold lower affinity than M32 (9.4 vs. 0.07 nM). In the same tissue, we also examined the effects of these peptides on adenylate cyclase activity. M32 showed a weak antagonistic behaviour but M242 acted as a potent biphasic regulator of adenylate cyclase. In conclusion, we present and characterise a new peptide M242, which could be a useful tool in studies of galaninergic signalling.