Comparison of Abeta levels in the brain of familial and sporadic Alzheimer's disease.

Mutations in presenilin (PS) and amyloid precursor protein (APP) genes are a predominant cause for early-onset familial Alzheimer disease (AD). Although these mutations are rare, they have in the past decades advanced our understanding of the underlying molecular mechanisms of AD. In the present study, Abeta levels were measured in cortical regions of APPsw and PS1 (M146V) mutation carriers, sporadic AD (SAD) and age-matched non-demented individuals. We found similar levels of soluble Abeta42, insoluble and soluble Abeta40 in both APPsw mutation carriers and SAD. However, lower levels of insoluble Abeta42 were detected in the frontal and temporal cortex of APPsw brain. In PS1 brain, insoluble Abeta40 and Abeta42 levels were significantly lower in all four cortical regions compared with SAD, whilst levels of Abeta40 were lower in frontal and occipital cortex compared with APPsw brain. The insoluble Abeta42/40 ratio was similar in SAD and APPsw but significantly higher in PS1 mutation carriers. Our results indicate that the pattern of Abeta deposition in PS1 mutation carriers differs from that in both APPsw and SAD, whereas the pattern in APPsw mutation carriers is more similar to that in SAD.

Nicotinic receptor agonists and antagonists increase sAPPalpha secretion and decrease Abeta levels in vitro.

We have earlier reported that Abeta were significantly reduced in brains of smoking Alzheimer patients and control subjects compared with non-smokers, as well as in nicotine treated APPsw transgenic mice. To examine the mechanisms by which nicotine modulates APP processing we here measured levels of secreted amyloid precursor protein (sAPPalpha), total sAPP, Abeta40 and Abeta42 in different cell lines expressing different nicotinic receptor (nAChR) subtypes or no nAChRs. Treatment with nicotine increased release of sAPPalpha and at the same time lowered Abeta levels in both SH-SY5Y and SH-SY5Y/APPsw cells expressing alpha3 and alpha7 nAChR subtypes. These effects could also be evoked by co-treatment with the competitive alpha7 nAChR antagonists alpha-bungarotoxin and methyllycaconitine (MLA), and by these antagonists alone, suggesting that binding to the agonist binding site, rather than activation of the receptor, may be sufficient to trigger changes in APP processing. The nicotine-induced increase in sAPPalpha could only be blocked by co-treatment with the open channel blocker mecamylamine. In addition to nicotine, the agonists epibatidine and cytisine both significantly increased the release of sAPP in M10 cells expressing the alpha4/beta2 nAChR subtype, and this effect was blocked by co-treatment with mecamylamine but not by the alpha4/beta2 competitive antagonist dihydro-beta-erythroidine. The lack of effect of nicotine on sAPPalpha and Abeta levels in HEK 293/APPsw cells, which do not express any nAChRs, demonstrates that the nicotine-induced attenuation of beta-amyloidosis is mediated by nAChRs and not by a direct effect of nicotine. Our data show that nicotinic compounds stimulate the non-amyloidogenic pathway and that alpha4 and alpha7 nAChRs play a major role in modulating this process. Nicotinic drugs directed towards specific nAChR subtypes might therefore be beneficial for the treatment of AD not only by lowering Abeta production but also by enhance release of neuroprotective sAPPalpha.

Age-dependent decline of neprilysin in Alzheimer's disease and normal brain: inverse correlation with A beta 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.

Changes in the activity and protein levels of CSF acetylcholinesterases in relation to cognitive function of patients with mild Alzheimer's disease following chronic donepezil treatment.

OBJECTIVES: To evaluate long-term changes in acetylcholinesterase (AChE) activity in CSF and blood following donepezil treatment in relation to the concentration of donepezil and cognition in AD patients. METHODS: CSF or blood (or both) samples of a total of 104 patients with mild AD were used [MMSE score 23 +/- 0.4; age 75 +/- 1 years (mean +/- SEM); n=53 for CSF and n=51 for plasma/red blood cell (RBC) samples]. The patients were treated with 5 or 10 mg/day donepezil and clinically followed for 2 years. The CSF and RBC AChE activities were measured by the Ellman's direct colorimetric assay. Protein levels of two variants of AChE ("read-through" AChE-R and synaptic AChE-S) were determined by an ELISA-like method. RESULTS: The plasma donepezil concentration was dose-dependent (between 30 and 60 ng/mL in the 5-mg and 10-mg group, respectively). The CSF donepezil concentration was 10 times lower than the plasma level and showed dose- and time-dependent kinetics. The RBC AChE inhibition was moderate (19-29%). CSF AChE-S inhibition was estimated to 30-40% in the 5-mg and 45-55% in the 10-mg group. Positive correlations were observed between the CSF AChE inhibition, an increased protein level of the AChE-R variant and MMSE examination. Patients with high AChE inhibition (>or=45%) showed a stabilized MMSE test result after up to two years, while a significant decline was observed in AD patients with lower AChE inhibition (

Smoking during early pregnancy affects the expression pattern of both nicotinic and muscarinic acetylcholine receptors in human first trimester brainstem and cerebellum.

Prenatal nicotine exposure is associated with an increased risk of complications during pregnancy and childhood. In this study the expression of nicotinic and muscarinic acetylcholine receptors in first trimester pons, medulla oblongata and cerebellum from abortus (5-12 weeks of gestation) of smoking and nonsmoking women was compared. A significant age-related increase in binding of nicotinic receptor subtype alpha4 was found in both pons and cerebellum only in fetal tissue from non-smoking women, while a similar increase was observed in medulla oblongata from fetuses exposed to smoking. A significant age-related increase in binding of muscarinic receptor subtype m2 was observed in pons from abortus of smoking compared with non-smoking women. The gene expression pattern of both alpha4 and alpha7 nicotinic receptor subunits was changed after smoking in all three regions investigated. Smoking also changed the expression of m1 and 2 muscarinic receptor mRNA in pons, m1 mRNA in cerebellum and the m3 mRNA in medulla oblongata. The findings indicate that early prenatal nicotine exposure affects the normal developmental pattern of the cholinergic system in human fetal brain.

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

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

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 Alzheimer's 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 ( approximately 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.

Protein and mRNA levels of nicotinic receptors in brain of tobacco using controls and patients with Alzheimer's disease.

The neuronal nicotinic receptors (nAChRs) are involved in several processes in brain including nicotine dependence and cognitive disorders. While the number of nAChRs in the brain of tobacco smokers is up-regulated, the receptors are reduced in the brain of patients with Alzheimer's disease (AD). The aim of this study was to investigate nAChR mRNA and protein levels in brain of smoking and non-smoking controls and AD patients. Western blotting and RT-PCR techniques were used to quantify different nAChR subunits in autopsy brain. The alpha4 and alpha7 but not the alpha3 nAChR protein levels were significantly increased in the temporal cortex of smoking (SC) compared with non-smoking controls (NSC). The alpha4-protein level was significantly higher in the temporal cortex of smoking AD (SAD) patients compared with non-smoking AD (NSAD). No changes in the alpha3, alpha4 or alpha7 subunits protein level were found in the hippocampus in any of the smoking groups. For both SADs and NSADs the protein levels for the alpha3 and alpha4 in temporal cortex and hippocampus and alpha7 in the hippocampus were significantly lower compared with non-smoking controls. No significant differences in alpha4 and alpha7 mRNA levels were detected in the hippocampus or temporal cortex of smokers compared with non-smokers. In conclusion this study showed an increased level of alpha4 and alpha7 nAChRs subunits in the temporal cortex of SC compared with NSC. This up-regulation was also seen in SAD although the protein levels of nAChR subunits were still lower in smoking AD brain compared with the NSC. The up-regulation of nAChRs in smoking groups and the loss of these receptors in AD patients were not correlated to any changes at the mRNA level suggesting that these changes may reflect post-transcriptional events.

Cerebral glucose metabolism, cerebrospinal fluid-beta-amyloid1-42 (CSF-Abeta42), tau and apolipoprotein E genotype in long-term rivastigmine and tacrine treated Alzheimer disease (AD) patients.

We evaluated cerebral glucose metabolism (CMRglc) and cerebrospinal fluid (CSF) levels of tau and beta-amyloid(1-42) (Abeta42), in relation to apolipoprotein E (ApoE) genotype, in patients with mild Alzheimer disease (AD) treated with rivastigmine (n=11) and tacrine (n=16) for 1 year; and two untreated AD groups. The rivastigmine-treated AD patients showed a significant increase in CMRglc as compared to both tacrine-treated and untreated AD subjects. The rivastigmine-treated AD group showed no change in CSF-tau levels after 1 year, while in contrast a significant increase as seen in tacrine-treated and untreated AD patients. The CSF-tau changes were mainly seen in ApoE epsilon4 carriers. There was no significant change in Abeta42 after 1-year treatment with either rivastigmine or tacrine. This study shows that the two long-term cholinesterase inhibitor treatments exert different effects on biological markers for AD.

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.

Nicotine-induced alterations in the expression of nicotinic receptors in primary cultures from human prenatal brain.

The nicotinic receptor proteins and gene transcripts for the different nicotinic receptor subunits exist in human prenatal brain already at 4-5 weeks of gestation. The early presence of nicotinic receptors suggests an important role for these receptors in modulating dendritic outgrowth, establishment of neuronal connections and synaptogenesis during development. When measurements of nicotinic receptors using [(3)H]epibatidine (labelling both the alpha3 and alpha4 subtype) and [(3)H]cytisine (labelling the alpha4 subtype) were performed in intact cells from the cortex, subcortical forebrain and mesencephalon (7.5-11 weeks of gestation), the highest specific binding for both ligands was detected in cells from mesencephalon, followed by subcortical forebrain and cortex. The effects of nicotine exposure were studied in primary cultures of prenatal brain (7.5-11 weeks of gestation). Treatment with nicotine (1-100 microM) for 3 days significantly increased the specific binding of [(3)H]epibatidine and [(3)H]cytisine in cortical cells but not in cells from subcortical forebrain and mesencephalon brain regions, indicating region-specific differences in the sensitivity to nicotine exposure. Relative quantification of mRNA showed that the expression of the nicotinic receptor subunits alpha3 and alpha7, but not alpha4, was increased in cortical cells after nicotine treatment. These findings support the assumption of a potential risk of disturbance in the functional role of nicotinic receptors during brain development as a consequence of maternal smoking during pregnancy.

Responder characteristics to a single oral dose of cholinesterase inhibitor: a double-blind placebo-controlled study with tacrine in Alzheimer patients.

A proportion of Alzheimer's disease (AD) patients treated for several months with cholinesterase (ChE) inhibitors have shown some favorable response on cognition, but the characteristics of the responders are still unclear. This study attempts to identify the characteristics of individuals with a positive behavioral response after a double-blind randomized administration of a single oral dose of tacrine (40 mg) and placebo to AD patients. Furthermore, the relationship between single-dose and long-term responders are examined. Twenty-four mildly to very mildly demented AD patients participated in the study. They all fulfilled the diagnosis of probable AD according to NINCDS-ADRDA criteria. Active treatment (tacrine 40 mg) and placebo was administered in random order on 2 consecutive days, and the effects were evaluated within 2 h using neuropsychological tests (assessing visuospatial ability, episodic memory and attention), registration of EEG activity and measurement of red blood cells (RBC) acetylcholinesterase (AChE), ChE activity and concentrations of tacrine and its metabolites in plasma. Results demonstrated significant improvement, tacrine compared to placebo, in measures of attention, but not in episodic memory or visuospatial ability. A single-dose response was therefore defined in terms of improvement in attention. The tacrine plasma concentration (pcTHA) showed a positively skewed distribution (mean +/- SD: 10.5 +/- 11.8, range: 1.0-51.8 ng/ml). There were no significant differences between single-dose responders compared to nonresponders in pcTHA, metabolites of tacrine, inhibition of AChE in RBC, tau levels in CSF, AChE activity in CSF or plasma and demographic variables. However, single-dose responders showed a higher right frontal alpha/theta ratio on EEG and had lower glucose metabolism in the parietal-temporal association cortex at baseline. In addition, the frequency of apolipoprotein E (APOE) epsilon 4 alleles was higher in responders. Interestingly, the single-dose response was related to the long-term response, although not significantly, which probably was due to lack of power. To conclude, the present study identified single-dose responders in terms of improved attentional performance associated with a relatively higher alpha/theta activity in the right frontal regions of the brain measured on EEG and predominance of APOE epsilon 4 allele.

Expression of nicotinic acetylcholine receptors in human and rat adrenal medulla.

Neuronal nicotinic receptors (nAChRs) are expressed in the brain but also in the peripheral tissues including the adrenal medulla. However, it is unclear which nAChRs are present in the human adrenal medulla. In the study, receptor binding assay, Western blot and RT-PCR have been performed to investigate the expression of nAChRs in adrenal medulla from human, rat and mouse. The results showed that in human adult adrenal medulla, mRNAs for nAChR alpha3, alpha4, alpha5, alpha7, beta2, beta3, and beta4 subunits but not beta2 in the fetal human adrenal medulla were expressed. Saturation binding of [3H]epibatidine showed two binding sites in human aged adrenal medulla. The specific binding of [3H]epibatidine (0.1 nM) was significantly higher in human fetal compared to human aged adrenal medulla. mRNAs for the alpha3, alpha4, alpha5, alpha7, beta2, and beta4 subunits but not the beta3 were detectable in adult rat and mouse adrenal medulla. No differences in gene-expression of the nAChRs were observed between new born, adult and aged rat adrenal medulla. Saturation binding of [3H]epibatidine showed only one binding site in rat adrenal medulla. Lower protein levels for the nAChR subunits were observed in the rat adrenal medulla compared to rat brain. There was lower protein levels of the nAChRs in aged rat adrenal medulla compared to the young rats. Sub-chronic treatment of nicotine to rats did not influence level of the nAChRs in the adrenal medulla. In conclusion, the expression of nAChRs in adrenal medulla is age- related and species dependent.

Nicotinic acetylcholine receptors during prenatal development and brain pathology in human aging.

Nicotinic acetylcholine receptor (nAChRs) proteins and gene transcripts are already present in human prenatal brain and spinal cord at 4-6 weeks gestation, and a clear age-related increase in number of nAChRs was apparent during first trimester. In pons, there was also a parallel increase in the alpha7 mRNA level with age. The highest specific binding of [3H]epibatidine and [3H]cytisine was detected in spinal cord, pons and medulla oblongata, and binding of [125I]alpha-bungarotoxin was highest in spinal cord, medulla oblongata and mesencephalon. From the late fetal stage brain nAChRs have been shown to fall with increasing age. During aging (between 40 and 100 years) high affinity nicotine binding in the frontal cortex decreases in parallel with glutamate NMDA receptor binding ([3H]MK801). In the hippocampal formation and entorhinal cortex nicotine binding also declines with age, in common with [125I]alpha-bungarotoxin in the entorhinal cortex, but NMDA receptor binding remains unchanged. These reductions in nicotine binding with age may predispose the neo- and archicortex to the loss of nAChRs observed in age-associated neurodegenerative conditions. By contrast no loss in nAChR binding with aging is observed in the thalamus and only after the 70th decade in the striatum, although in Alzheimer's disease, Parkinson's disease and Lewy body dementia deficits in nAChRs are observed in these areas and may be associated with specific disease-related processes.

Modulation of beta-amyloid precursor protein processing and tau phosphorylation by acetylcholine receptors.

Neurofibrillary lesions and senile plaques that are composed mainly of hyperphosphorylated tau protein and the amyloid-beta peptide derived from the amyloid precursor protein, respectively, are classical hallmarks of Alzheimer's disease. A number of studies strongly suggests that amyloid-beta formation and amyloid depositions are linked to the pathogenesis of Alzheimer's disease. Recent findings suggest that very low concentrations of the amyloid-beta can inhibit various cholinergic neurotransmitter functions independently of apparent neurotoxicity. Many factors have been shown to influence the processing of amyloid precursor protein, including activation of muscarinic and nicotinic receptors. This review focus on some recent studies concerning the regulation of amyloid precursor protein processing and modulation of tau phosphorylation by acetylcholine receptor stimulation and how cholinergic deficits and amyloid-beta might be related to one another.

Increased levels of tau protein in SH-SY5Y cells after treatment with cholinesterase inhibitors and nicotinic agonists.

Several cholinesterase inhibitors used in the treatment of Alzheimer's disease (AD) have been shown to interact with an allosteric site on the nicotinic acetylcholine receptor (nAChR). A possible linkage between the phosphorylation state of tau, the major component of paired helical filaments found in AD brain, and stimulation of nAChRs by cholinesterase inhibitors and nicotinic agonists was investigated. Western blot analysis showed that treatment of SH-SY5Y cells for 72 h with the cholinesterase inhibitors tacrine (10(-5) M), donepezil (10(-5) M), and galanthamine (10(-5) M), nicotine (10(-5) M), and epibatidine (10(-7) M) increased tau levels as detected with Tau-1, AT 8, and AT 270 monoclonal antibodies and binding of [3H]epibatidine. The increase in tau immunoreactivity induced by nicotine, epibatidine, and tacrine, but not the up-regulation of nAChRs, was prevented by the antagonists d-tubocurarine and mecamylamine. Both antagonists were synergistic with the nicotinic agonists in causing up-regulation, but only d-tubocurarine showed a synergistic effect with tacrine. The increased tau immunoreactivity induced by tacrine was not prevented by atropine, indicating that in terms of cholinergic receptors, tacrine modulates tau levels mainly through interactions with nAChRs and not with muscarinic receptors. Additional work is needed to determine the exact mechanism by which cholinesterase inhibitors and nicotinic agonists modulate phosphorylation and levels of tau protein.

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 Alzheimer's 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.

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.

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.

Regulation of nicotinic receptor subtypes following chronic nicotinic agonist exposure in M10 and SH-SY5Y neuroblastoma cells.

The present study further investigated whether nicotinic acetylcholine receptor (nAChR) subtypes differ in their ability to up-regulate following chronic exposure to nicotinic agonists. Seven nicotinic agonists were studied for their ability to influence the number of chick alpha4beta2 nAChR binding sites stably transfected in fibroblasts (M10 cells) following 3 days of exposure. The result showed a positive correlation between the Ki values for binding inhibition and EC50 values for agonist-induced alpha4beta2 nAChR up-regulation. The effects of epibatidine and nicotine were further investigated in human neuroblastoma SH-SY5Y cells (expressing alpha3, alpha5, beta2, and beta4 nAChR subunits). Nicotine exhibited a 14 times lower affinity for the nAChRs in SH-SY5Y cells as compared with M10 cells, whereas epibatidine showed similar affinities for the nAChRs expressed in the two cell lines. The nicotine-induced up-regulation of nAChR binding sites in SH-SY5Y cells was shifted to the right by two orders of magnitude as compared with that in M10 cells. The epibatidine-induced up-regulation of nAChR binding sites in SH-SY5Y cells was one-fourth that in M10 cells. The levels of mRNA of the various nAChR subunits were measured following the nicotinic agonist exposure. In summary, the various nAChR subtypes show different properties in their response to chronic stimulation.