We investigated the functional characteristics of pre- and postsynaptic cholinergic transmission in APPswe/PS1dE9 double transgenic mice at a young age (7-10 weeks) before the onset of amyloid plaque formation and at adult age (5-6 months) at its onset. We compared brain slices from cerebral cortex and hippocampus with amyloid deposits to slices from striatum with no amyloid plaques by 6 months of age. In young transgenic mice we found no impairments of preformed and newly synthesized [(3)H]-ACh release, indicating intact releasing machinery and release turnover, respectively. Adult transgenic mice displayed a significant increase in preformed [(3)H]-ACh release in cortex but a decrease in hippocampus and striatum. The extent of presynaptic muscarinic autoregulation was unchanged. Evoked release of newly synthesized [(3)H]-ACh was significantly reduced in the cortex and hippocampus but unchanged in the striatum. Carbachol-induced G-protein activation in cortical membranes displayed decreased potency but normal efficacy in adult animals and no changes in young animals. These results indicate that functional pre- and postsynaptic cholinergic deficits are not present in APPswe/PS1dE9 transgenic mice before 10 weeks of age, but develop along with beta-amyloid accumulation in the brain.
- MeSH
- Acetylcholine deficiency MeSH
- Alzheimer Disease genetics metabolism physiopathology MeSH
- Amyloid metabolism MeSH
- Amyloid beta-Protein Precursor genetics MeSH
- Cholinergic Agonists pharmacology MeSH
- Cholinergic Fibers metabolism pathology MeSH
- Nerve Degeneration metabolism pathology MeSH
- Down-Regulation genetics MeSH
- Hippocampus metabolism pathology physiopathology MeSH
- Disease Models, Animal MeSH
- Brain Chemistry genetics MeSH
- Brain growth & development metabolism physiopathology MeSH
- Cerebral Cortex growth & development metabolism physiopathology MeSH
- Mice, Transgenic MeSH
- Mice MeSH
- Organ Culture Techniques MeSH
- Presenilin-1 genetics MeSH
- GTP-Binding Proteins drug effects genetics metabolism MeSH
- Receptors, Muscarinic metabolism MeSH
- Aging metabolism MeSH
- Age Factors MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Choline is an essential nutrient necessary for synthesis of membrane phospholipids, cell signalling molecules and acetylcholine. The aim of this study was to detect and characterize the choline transporter-like 1 (CTL1/SLC44A1) protein in CNS tissues and the hybrid neuroblastoma x glioma cell line NG108-15, which synthesizes acetylcholine and has high affinity choline transport but does not express the cholinergic high affinity choline transporter 1. The presence of CTL1 protein in NG108-15 cells was confirmed using our antibody G103 which recognizes the C-terminal domain of human CTL1. Three different cognate small interfering RNAs were used to decrease CTL1 mRNA in NG108-15 cells, causing lowered CTL1 protein expression, choline uptake and cell growth. None of the small interfering RNAs influenced carnitine transport, demonstrating the absence of major non-specific effects. In parental C6 cells knockdown of CTL1 also reduced high affinity choline transport. Our results support the concept that CTL1 protein is necessary for the high affinity choline transport which supplies choline for cell growth. The presence of CTL1 protein in rat and human CNS regions, where it is found in neuronal, glial and endothelial cells, suggests that malfunction of this transporter could have important implications in nervous system development and repair following injury, and in neurodegenerative diseases.
- MeSH
- Acetylcholine biosynthesis MeSH
- Cell Differentiation physiology MeSH
- Cell Membrane chemistry metabolism MeSH
- Antigens, CD chemistry immunology metabolism MeSH
- Choline metabolism MeSH
- Down-Regulation genetics MeSH
- Glioma MeSH
- Hybridomas MeSH
- Immunohistochemistry MeSH
- Rats MeSH
- Humans MeSH
- RNA, Small Interfering physiology MeSH
- RNA, Messenger metabolism MeSH
- Cell Line, Tumor MeSH
- Neuroblastoma MeSH
- Neurogenesis physiology MeSH
- Neurons metabolism MeSH
- Organic Cation Transport Proteins chemistry immunology metabolism MeSH
- Antibody Specificity MeSH
- Central Nervous System Stimulants metabolism MeSH
- Protein Structure, Tertiary physiology MeSH
- Cell Enlargement MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Animals MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
We assessed the integrity of cholinergic neurotransmission in parietal cortex of young adult (7 months) and aged (17 months) transgenic APPswe/PS1dE9 female mice compared to littermate controls. Choline acetyltransferase and acetylcholinesterase activity declined age-dependently in both genotypes, whereas both age- and genotype-dependent decline was found in butyrylcholinesterase activity, vesicular acetylcholine transporter density, muscarinic receptors and carbachol stimulated binding of GTP gamma S in membranes as a functional indicator of muscarinic receptor coupling to G-proteins. Notably, vesicular acetylcholine transporter levels and muscarinic receptor-G-protein coupling were impaired in transgenic mice already at the age of 7 months compared to wild type littermates. Thus, brain amyloid accumulation in this mouse model is accompanied by a serious deterioration of muscarinic transmission already before the mice manifest significant cognitive deficits.
- MeSH
- Acetylcholinesterase metabolism MeSH
- Amyloid beta-Protein Precursor metabolism MeSH
- Analysis of Variance MeSH
- Financing, Organized MeSH
- Animals, Genetically Modified MeSH
- Guanosine 5'-O-(3-Thiotriphosphate) metabolism MeSH
- Humans MeSH
- Mutation MeSH
- Mice MeSH
- N-Methylscopolamine MeSH
- Synaptic Transmission genetics MeSH
- Piperidines pharmacokinetics metabolism MeSH
- Presenilin-1 genetics MeSH
- Receptors, Muscarinic metabolism MeSH
- Protein Binding drug effects MeSH
- Age Factors MeSH
- Vesicular Acetylcholine Transport Proteins metabolism MeSH
- Dose-Response Relationship, Drug MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
We studied the effects of 3-[3-hexyloxy-1,2,5-thiadiazo-4-yl]-1,2,5,6-tetrahydro-1-methylpyridine (xanomeline) wash-resistant binding on presynaptic muscarinic regulation of electrically evoked [(3)H]acetylcholine (ACh) release from rat brain slices. In both cortical and striatal tissues that possess M(2) and M(4) autoreceptors, respectively, immediate application of 10 microM xanomeline had no effect on evoked [(3)H]ACh release or its inhibition by 10 microM carbachol. In contrast, preincubation with 1, 10, or 100 microM xanomeline for 15 min decreased evoked release of ACh measured after 53 min of washing in xanomeline-free medium in a concentration-dependent manner. The maximal inhibitory effect equaled the immediate effect of the muscarinic full agonist carbachol, and it was completely (at 1 and 10 microM xanomeline) or partially (at 100 microM xanomeline) blocked by 1 microM N-methylscopolamine. Neither presence of N-methylscopolamine during 100 microM xanomeline treatment nor previous irreversible inactivation of the classical receptor binding site using propylbenzylcholine mustard in cortical slices prevented the inhibitory effect of wash-resistantly bound xanomeline. Treatment of cortical slices with xanomeline slightly decreased the number of muscarinic binding sites, and it markedly decreased affinity for N-methylscopolamine. When applied as in acetylcholine release experiments, xanomeline did not impair presynaptic alpha(2)-adrenoceptor-mediated regulation of noradrenaline release. The functional studies in brain tissue reported in this work demonstrate that xanomeline can function as a wash-resistant agonist of native presynaptic muscarinic M(2) and M(4) receptors with both competitive and allosteric components of action.
- MeSH
- Acetylcholine secretion MeSH
- Muscarinic Agonists metabolism MeSH
- CHO Cells MeSH
- Financing, Organized MeSH
- Cricetinae MeSH
- Rats MeSH
- Humans MeSH
- Cerebral Cortex secretion drug effects MeSH
- Norepinephrine secretion MeSH
- Rats, Wistar MeSH
- Pyridines pharmacology MeSH
- Receptor, Muscarinic M2 physiology drug effects MeSH
- Receptor, Muscarinic M4 physiology drug effects MeSH
- Thiadiazoles pharmacology MeSH
- Animals MeSH
- Check Tag
- Cricetinae MeSH
- Rats MeSH
- Humans MeSH
- Male MeSH
- Animals MeSH
