Ewa Hellström-Lindahl

Regional distribution of nicotinic receptor subunit mRNAs in human brain : comparison between Alzheimer and normal brain

The regional expression of mRNA for the nicotinic acetylcholine receptor (nAChR) subunits alpha3, alpha4 and alpha7 was examined in postmortem brain tissues from controls and patients with Alzheimers disease (AD) by using quantitative RT-PCR. In parallel, the numbers of nAChRs were measured by receptor binding. Relative quantification of the nAChR gene transcripts in control brains showed that expression of alpha3 was highest in the parietal cortex, frontal cortex and hippocampus, and lower in the temporal cortex and cerebellum. The highest level of alpha4 mRNA was found in the temporal cortex and cerebellum, while alpha7 mRNA was equally distributed in all brain regions except for hippocampus where it was less abundant. In comparison with AD brains, no differences in the expression of alpha3 and alpha4 in the temporal cortex, hippocampus and cerebellum were found. The level of alpha7 mRNA was significantly higher in the hippocampus of AD brains compared to controls. The binding sites for [3H] epibatidine and [3H] nicotine in the temporal cortex and [125I] alpha-bungarotoxin in hippocampus were significantly decreased in AD patients compared to controls. Saturation analysis of [3H] epibatidine binding revealed two classes of binding sites, with a significant reduction of the higher affinity epibatidine binding sites in the temporal cortex of AD brain. The results show that there is a regional distribution of the expression of the different nAChRs subunits in human brain. The findings that the alpha3 and alpha4 mRNA levels were not changed in AD brains suggest that the loss of higher affinity epibatidine binding sites observed in AD patients cannot be attributed to alternations at the transcriptional level of the alpha3 and alpha4 genes and that causes have to be searched for at the translational and/or posttranslational level. The increased mRNA level of alpha7 previously found in lymphocytes, and now also in the hippocampus of AD patients, indicate that subunit specific changes in gene expression of nAChRs is associated with AD.

Chronic nicotine treatment reduces β-amyloidosis in the brain of a mouse model of Alzheimer's disease (APPsw)

Alzheimers disease neuropathology is characterised by β‐amyloid plaques and neurofibrillary tangles. Inhibition of β‐amyloid accumulation may be essential for effective therapy in Alzheimers disease. In this study we have treated transgenic mice carrying the Swedish mutation of human amyloid precursor protein [Tg(Hu.APP695.K670N‐M671L)2576], which develop brain β‐amyloid deposits, with nicotine in drinking fluid (200 µg/mL) from 9–14.5 months of age (5.5 months). A significant reduction in amyloid β peptide 1–42 positive plaques by more than 80% (p < 0.03) was observed in the brains of nicotine treated compared to sucrose treated transgenic mice. In addition, there was a selective reduction in extractable amyloid β peptides in nicotine treated mice; cortical insoluble 1–40 and 1–42 peptide levels were lower by 48 and 60%, respectively (p < 0.005), whilst there was no significant change in soluble 1–40 or 1–42 levels. The expression of glial fibrillary acidic protein was not affected by nicotine treatment. These results indicate that nicotine may effectively reduce amyloid β peptide aggregation in brain and that nicotinic drug treatment may be a novel protective therapy in Alzheimers disease.

REGIONAL DISTRIBUTION OF NICOTINIC RECEPTORS DURING PRENATAL DEVELOPMENT OF HUMAN BRAIN AND SPINAL CORD

The development of nicotinic acetylcholine receptors (nAChRs) in brains from human fetuses of 4-12 weeks gestational age was studied. The expression of nAChR subunit mRNAs was analyzed using reverse transcriptase-polymerase chain reaction. Expression of alpha 3, alpha 4, alpha 5, alpha 7, beta 2, beta 3 and beta 4 mRNA were all detected in the prenatal spinal cord, medulla oblongata, pons, cerebellum, mesencephalon, subcortical forebrain and cortex during first trimester development. Relative quantification of mRNA showed that the highest levels for alpha 3, alpha 4 and alpha 7 were expressed in the spinal cord, alpha 5 was most abundant in the cortex and beta 3 was highest in the cerebellum. beta 4 seemed to be equally distributed in all regions whereas beta 2 was high in the cortex and cerebellum. A comparison of expression of nAChR subunit mRNAs in the cortex and cerebellum of prenatal and aged (54-81 years) brain showed that mRNA levels for alpha 4, alpha 5, alpha 7, beta 2 and beta 4 were significantly higher in the prenatal cortex and cerebellum than in aged brain, whereas the level of alpha 3 transcript was similar, and beta 3 significantly higher in aged cortex. Specific binding of [3H]-epibatidine to prenatal brain membranes was detected as early as 4-5 weeks of gestation in the spinal cord, medulla oblongata, pons and subcortical forebrain. A positive correlation between gestational age and [3H]-epibatidine and [3H]-cytisine binding was found in several brain regions. The highest specific binding of [3H]-epibatidine and [3H]-cytisine was detected in the spinal cord, pons and medulla oblongata and the lowest in the cortex. Saturation analysis of [3H]-cytisine binding in both prenatal and aged brain were best fit by a model for a single site, whereas binding data for [3H]-epibatidine revealed two classes of binding sites. The early presence of nAChR proteins and gene transcripts shown in the present study suggests an important role for nAChRs in modulating dendritic outgrowth, establishment of neuronal connections and synaptogenesis during development.

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. MethodsEleven 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. ResultsAt 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. ConclusionsRivastigmine 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.

Age-dependent decline of neprilysin in Alzheimer's disease and normal brain: Inverse correlation with Aβ levels

Brain deposition of amyloid-beta (A beta) is a pathological hallmark of Alzheimer disease (AD) but A beta is also detected in non-demented elderly individuals. Neprilysin has been shown to be an important enzyme to degrade A beta in brain. We investigated whether decreased neprilysin levels contributes to the accumulation of A beta in AD and in normal aging. No difference in neprilysin protein and mRNA levels were found between AD subjects and age-matched controls. Protein levels of neprilysin were reduced with age in the temporal and frontal cortex of AD and normal brain. A significant positive correlation between insoluble A beta 40 and A beta 42 with age was found in cortex of normal brain whereas in AD brain the correlation between age and A beta was weaker. Our findings of an inverse correlation between neprilysin and insoluble A beta levels in both groups suggest that neprilysin is involved in the clearance of A beta. The observed age-dependent decline in neprilysin may be related to the increased A beta levels during normal aging. The similar rate of decline in neprilysin with age may not be the major cause of the high levels of A beta associated with AD but is likely to be a trigger of AD pathology.

Imaging of Nicotinic and Muscarinic Receptors in Alzheimer's Disease: Effect of Tacrine Treatment

Functional imaging techniques offer new possibilities for further understanding of changes in functional correlates of structural and biological changes in dementia disorders like Alzheimers disease (AD). Regional disturbances in glucose metabolism and cerebral blood flow are known to occur in AD brains and probably roughly correlate to changes in neurotransmitter activities. A proper estimate would be to visualize the neuroreceptors themselves. In this study the cholinergic nicotinic and muscarinic receptors were studied in brain by positron emission tomography (PET). The rate constant k2* (s) (-)11C-nicotine was significantly higher (+43%) in temporal cortex of AD patients compared to controls (p < 0.017) indicating a lower binding of 11C-nicotine in AD brains compared to controls. Treatment with the cholinesterase inhibitor tacrine (80 mg daily) during 3 months to AD patients resulted in a mean plasma concentration of 7.7 +/- 0.8 ng/ml and a corresponding inhibition of the cholinesterase activity in plasma by 34 +/- 5%. A significantly lower k2* (increased binding) for 11C-nicotine binding (-15%; p < 0.006) was obtained in the temporal cortex after 3 months of treatment compared to prior treatment. The muscarinic antagonist 11C-benztropine was used to visualize muscarinic receptors and the binding capacity of 11C-benztropine (KR) was found to be decreased in the temporal cortex after 3 months of tacrine treatment.

Long-lasting acetylcholinesterase splice variations in anticholinesterase-treated Alzheimer's disease patients

Protein levels of different acetylcholinesterase (AChE) splice variants were explored by a combination of immunoblot techniques, using two different antibodies, directed against the C‐terminus of the AChE‐R splice variant or the core domain common to all variants. Both AChE‐R and AChE‐S splice variants as well as several heavier AChE complexes were detected in brain homogenates from the parietal cortex of patients with or without Alzheimers disease (AD) as well as the cerebrospinal fluid (CSF) of AD patients, compatible with the assumption that CSF AChEs might originate from CNS neurons. Long‐term changes in the composition of CSF AChE variants were further pursued in AD patients treated with rivastigmine (n = 11) or tacrine (n = 17) in comparison to untreated AD patients (n = 5). In untreated patients, AChE‐R was markedly reduced as compared with the baseline level (37%), whereas the medium size AChE‐S complex was increased by 32%. Intriguingly, tacrine produced a general and profound up‐regulation of all detected AChE variants (up to 117%), whereas rivastigmine treatment caused a mild and selective up‐regulation of AChE‐R (∼10%, p < 0.05). Moreover, the change in the ratio of AChE‐R to AChE‐S (R/S‐ratio) strongly and positively correlated with sustained cognition at 12 months (p < 0.0001). Thus, evaluation of changes in the composition of CSF AChE variants may yield important information referring to the therapeutic efficacy and/or development of drug tolerance in AD patients treated with anti‐cholinesterases.

Smoking during pregnancy: a way to transfer the addiction to the next generation?

Many epidemiological studies support a relationship between maternal smoking during pregnancy and adverse neurobehavioral effects later in life. Prenatal exposure to tobacco seems to increase the risks for cognitive deficits, attention deficit/hyperactivity disorder, conduct disorder, criminality in adulthood and a predisposition in the offspring to start smoking and alcohol abuse. Nicotine readily crosses the placenta and the fetuses of mothers who smoke are exposed to relatively higher nicotine concentrations than their mothers. In the fetal brain nicotine can activate nicotinic receptors which play an important role during development of the brain. A direct specific action on the developing human brain is plausible during the major part of the prenatal life, since the nicotinic receptors are already present in the brain during the first trimester.

Long-term tacrine treatment in three mild Alzheimer patients: effects on nicotinic receptors, cerebral blood flow, glucose metabolism, EEG, and cognitive abilities.

The effect of long-term treatment with tacrine (tetrahydroaminoacridine) was studied in three Alzheimer patients (aged 57, 64, and 68 years) with mild dementia. All three patients had a Mini-Mental State Examination score of 24/30 and carried at least one apolipoprotein E (ApoE) epsilon4 allele. Tacrine was given in doses between 80 and to 160 mg daily for 13-31 months. A lower tacrine concentration was observed generally in cerebrospinal fluid (CSF) compared with plasma. The acetylcholinesterase activity in CSF tended to be increased following longer periods of tacrine treatment, whereas the butyrylcholinesterase activity was decreased. The three patients repeatedly underwent positron emission tomography investigation of cerebral blood flow, nicotinic receptors, cerebral glucose metabolism, and electroencephalogram (EEG) and cognitive tests. Positive influences on these parameters were observed following both short-term and long-term treatment with tacrine. Improvement of nicotinic receptors (measured as 11C-nicotine binding), cerebral blood flow, EEG, and some cognitive tests (trail making test, block design test) occurred earlier after initiation of tacrine treatment compared with the glucose metabolism, which was increased after several months of tacrine treatment. An improvement in attention (trail making test) was observed following tacrine as sign for frontal lobe activation (EEG). The functional effects of tacrine in Alzheimer patients appeared to be related to both dose and length of cholinesterase inhibitor treatment.

Reduced levels of Aβ 40 and Aβ 42 in brains of smoking controls and Alzheimer's patients

The effects of nicotine on levels of Aβ 40 and Aβ 42 and nicotinic receptor binding sites were studied in brains from nonsmoking and smoking patients with Alzheimers disease (AD) and aged-matched controls. The levels of soluble and insoluble Aβ 40 and Aβ 42 in frontal cortex and Aβ 40 in temporal cortex and hippocampus were significantly decreased in smoking AD patients compared to nonsmokers with AD. In smoking controls the levels of soluble and insoluble Aβ 40 and Aβ 42 in the frontal and temporal cortex were significantly lower than in nonsmoking controls. The binding of [3H]cytisine in temporal cortex was significantly increased in smokers with AD compared to nonsmokers with AD. In smoking controls [3H]cytisine and [3H]epibatidine binding were significantly increased from 1.5- to 2-fold compared to nonsmoking controls whereas binding sites for [125I]α-bungarotoxin was less up-regulated. These results indicate that selective nicotinic receptor agonists may be a novel protective therapy in AD by reducing Aβ levels as well as the loss of nicotinic receptors in AD brain.