O5‐06‐06: LIGANDS OF THE CORE ACETYLCHOLINE BIOSYNTHESIZING ENZYME, CHOLINE ACETYLTRANSFERASE, AS NOVEL AND POTENTIAL THERAPEUTIC AGENTS AND IN VIVO PET TRACERS FOR EARLY DIAGNOSIS OF ALZHEIMER'S DISEASE
Taher Darreh‐ShoriRajnish KumarAmit KumarPrashant R. MurumkarSangram NagZhisheng JiaRyosuke ArakawaAntoine LeuzyLaëtitia LemoineAgneta NordbergMange Ram YadavChrister HalldinBengt Långström
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A key feature of Alzheimer's disease (AD) is an early and severe degeneration of the cholinergic projections throughout the brain. This characteristic is also shared by Lewy Body Dementia, Parkinson's disease dementia and Down's syndrome. Expression of the core acetylcholine (ACh) biosynthesizing enzyme, choline acetyltransferase (ChAT) defines this widespread neuronal system. Thereby ChAT was chosen as a prominent target for developing new in vivo probes for mapping cholinergic network, and designing novel cholinergic enhancing therapeutic agents. Advanced in silico molecular modelling analysis was used to screen diverse drug registries and an in-house molecular library against ChAT. The in silico hits were then examined with a robust in-house invented high-throughput ChAT assays using human recombinant protein and postmortem tissue extracts of 3 different brain regions of a group of AD (n=6) and controls (n=6). All hits were also tested against ACh degrading enzymes, AChE and BChE. We identified two different class of ChAT ligands. One class acted as highly potent inhibitor of ChAT, with low nanomolar activity. They show negligible activity against ACh degrading enzymes, indicating high selectivity for ChAT. The second class, on the contrary enhanced the intrinsic catalytic rate of ACh-biosynthesis by ChAT. These compounds compose the first ever identified small molecules, and are termed by us as ChAT Potentiating Ligands (CPLs). Systematic enzymes kinetic analyses identified 10 CPLs with EC50 values in low micromolar ranges. Some of CPLs also acted on AChE and/or BChE but as inhibitors. These are hence expected to exhibit a potentially dual mode of cholinergic enhancing actions of both increasing the biosynthesis of ACh, and inhibiting its degradation. Given the early and selective degeneration of the central cholinergic neurons in the major dementia disorders and ChAT being the defining marker of this neuronal system, the high affinity and selective ChAT inhibitors are being tested as ChAT-PET tracer. The CPLs on the other hand constitute a novel class of compounds of cholinergic enhancing agents that act by increasing biosynthesis of acetylcholine rather than preventing its degradation. They are hence expected to show superior efficacy than the currently in use cholinesterase inhibitors.The development of cholinergic neurons in mouse frontal cortex was studied both in vivo and in vitro by immunocytochemistry with an antibody to choline acetyltransferase (ChAT), the enzyme responsible for acetylcholine synthesis. While cortical cholinergic neurons have previously been characterized in rat cortex, up until very recently, intrinsic cortical cholinergic neurons were considered to be absent in mouse, and little is known about their development or phenotypic characteristics. The present study found no ChAT-positive neurons in mouse frontal cortex on postnatal day 0 (P0, the day of birth). On P7 there were few, faintly stained, ChAT-positive neurons. The numerical density of ChAT-positive neurons increased substantially with age, from none on P0, to 9.2 + 1.4 on P7, to 14.8 + 0.9 on P16, and 41.6 + 3.9 in adulthood. Considering that the numerical density of total neurons decreases during this postnatal period, the data represent a marked developmental increase in the percentage of cholinergic neurons. The development of cholinergic neurons showed very similar timelines in rat and mouse frontal cortex. Cultures prepared from mouse frontal cortex on embryonic day 16 were maintained for 25, 76, or 100 days in vitro (div). The percentage of ChAT-positive neurons was considerably higher than in vivo, ranging from a mean 28% to 31% across the three age (div) groups. With increasing age of the cultures, the numerical density of total neurons and ChAT-positive neurons decreased while the percentage of ChAT-positive neurons did not change significantly. These observations suggest some temporal stability in the cultures. Using dual immunofluorescence, ChAT-positive neurons were tested for colocalization with GAD or TH. The majority of ChAT-positive neurons colocalized with GAD, both in vitro and in vivo. However, ChAT did not colocalize with TH, either in vitro or in vivo. Our comparison of intact frontal cortex and cultures suggest that while the percentage of cholinergic neurons was greater in the cultures, the cholinergic neurons developed phenotypic similarities in vitro and in vivo.
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It is generally agreed that hippocampal acetylcholine (ACh) is synthesized and released exclusively from the terminals of the long-axon afferents whose cell bodies reside in the medial septum and diagonal band. The search for intrinsic cholinergic neurons in the hippocampus has a long history; however evidence for the existence of these neurons has been inconsistent, with most investigators failing to detect them using in situ hybridization or immunohistochemical staining of the cholinergic markers, choline acetyltransferase (CHAT) or vesicular acetylcholine transporter (VACHT). Advances in the use of bacterial artificial chromosome (BAC) transgenic mice expressing a reporter protein under the control of the genomic elements of the Chat gene (Chat-BAC mice) have facilitated studies of cholinergic neurons. Such mice show robust and faithful expression of the reporter proteins in all known cholinergic cell populations. The availability of the Chat-BAC mice re-ignited interest in hippocampal cholinergic interneurons, because a small number of such reporter-expressing cells is frequently observed in the hippocampus of these mice. However, to date, attempts to confirm that these neurons co-express the endogenous cholinergic markers CHAT or VACHT, or release ACh, have been unsuccessful. Without such confirmatory evidence it is best to conclude that there are no cholinergic neurons in the hippocampus. Similar considerations apply to other BAC transgenic lines, whose utility as a discovery tool for cell populations heretofore not known to express the genes of interest encoded by the BACs, must be validated by methods that detect expression of the endogenous genes.
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Background The recent availability of antisera to the vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT) that demonstrate peripheral cholinergic neurons has made possible the anatomical identification of cholinergic neurons in the enteric nervous system. In this study, we localised cholinergic neurons in the mouse small and large intestine and identified which substances are found colocalised in the cholinergic neurons. Methods Immunohistochemical single and double staining techniques were used on whole mount preparations and frozen sections to examine the localisation and chemical coding of cholinergic neurons in the small and large intestine of the mouse. Cholinergic neurons were identified using antisera to ChAT or VAChT. Results In both the small and large intestine, numerous ChAT-immunoreactive nerve cell bodies were present in the myenteric and submucous ganglia, and ChAT- and VAChT-immunoreactive nerve terminals were abundant in the myenteric and submucous plexuses and the external muscle. Previous studies have identified two major classes of myenteric neurons in the small intestine of the mouse—those containing calretinin plus substance P, and those containing nitric oxide synthase (NOS) plus vasoactive intestinal peptide (VIP). Double-label studies showed that the vast majority of the calretinin/substance P neurons were cholinergic neurons, whereas only a small proportion of the NOS/VIP cells were cholinergic; the noncholinergic NOS/VIP neurons were motor neurons or interneurons, whereas the cholinergic NOS/VIP neurons appeared to be exclusively interneurons. In the small intestine, all of the 5-HT–loaded neurons and a subpopulation of the calbindin neurons were also cholinergic. In the large intestine, there was a pattern of overlaps similar to that found in the small intestine, except that in the large intestine approximately 25% of the calretinin cells were not cholinergic. Only approximately one third of the GABA-loaded neurons in the large intestine were cholinergic. Conclusions Large subpopulations of motor neurons and interneurons in the mouse small intestine are cholinergic neurons. Anat. Rec. 251:185–199, 1998. © 1998 Wiley-Liss, Inc.
Vesicular acetylcholine transporter
Calretinin
Enteric Nervous System
Submucous plexus
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Forebrain
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In the neocortices and amygdalae of young and aged macaques, cholinergic axons were identified by means of a monoclonal antibody to bovine choline acetyltransferase. Many fine, linear, immunoreactive profiles were seen in these animals. In the older animals, some cholinergic axons showed multifocal enlargements along their course. In some instances, neurites with choline acetyltransferase immunoreactivity were associated with deposits of amyloid (visualized with thioflavin T fluorescence). The appearance of these amyloid-associated abnormal cholinergic processes was similar to that of neurites in senile plaques, as shown by conventional silver impregnation techniques. Cholinergic systems thus give rise to some of the neurites within senile plaques.
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Abstract Background Basal forebrain cholinergic dysfunction, likely linked to tau aggregation pathology, is a characteristic feature of AD. Cholinergic neurons contain choline acetyltransferase (ChAT) and high‐affinity tropomyosin‐related kinase A (TrkA) and send efferents to cortex and hippocampus where they release acetylcholine (ACh). The vesicular acetylcholine transporter (VAChT) is responsible for loading ACh into secretory vesicles and acetylcholine esterase (AChE) hydrolyses ACh in the synaptic cleft. We here aimed to evaluate the cholinergic phenotype in the Line 1 animal model of tauopathy and to determine the effect of the choline esterase inhibitor rivastigmine alone and in conjunction with the tau aggregation inhibitor hydromethylthionine on the cholinergic system. Method Line 1 (L1) and control NMRI mice, 8‐9 months old, divided into 13 groups (n = 5 each), were treated with rivastigmine (0.1 and 0.5 mg/kg) and hydromethylthionine (5 and 15 mg/kg) and their combinations for 11 weeks. Immunohistochemical staining in brain sections was performed for ChAT, TrkA, VAChT and tau with a repeat domain monoclonal antibody (TauRx Therapeutics Ltd.). AChE activity was measured histochemically. The state of the cholinergic system was determined by stereological counting of ChAT‐ir and TrkA‐ir neurons, while Relative Optical Intensity (ROI) was used to tau immunoreactivity in basal forebrain, and cholinergic projections were evaluated using VAChT and AChE ROI. Result Number of ChAT‐ir, TrkA‐ir neurons and ROI value for VAChT and AChE were significantly lower while ROI value for tau was higher in vehicle‐treated L1 mice compared with wild type control. Numbers of ChAT‐ir, TrkA‐ir neurons and ROI value of VAChT and AChE in L1 mice were increased and tau ROI was decreased by hydromethylthionine treatment. Combined treatment decreased numbers of ChAT‐ir, TrkA‐ir neurons and ROI for VAChT and AChE compared to L1 groups treated with hydromethylthionine alone. Conclusion There was a significant loss of cholinergic basal forebrain neurones, impaired cholinergic projection and increased tau staining in L1 mice. Monotherapy with hydromethylthionine improved the cholinergic neurons phenotype, enhanced their projection and reduced tau pathology. Combination therapy interacted negatively attenuating the effect of hydromethylthionine given alone.
Vesicular acetylcholine transporter
Nucleus basalis
Tau protein
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Alzheimer's disease (AD) is characterized by an extensive degeneration of the cholinergic system of the human septo-hippocampal pathway (Collerton, 1986) which is correlated with severe deficits of cognitive functions (Perry et al., 1978). It has been well established that in AD there are decreases of cholinergic function and cholinergic markers associated with this pathway. These deficits include the loss of cholinergic cell bodies in the basal forebain (Whitehouse et al., 1982; Arendt et al., 1983) and decreased choline acetyltransferase (ChAT) activity in the basal forebrain and hippocampal formation (Perry et al., 1978; Araujo et al., 1988). In addition, the ability of hippocampal cholinergic neurons to synthesize acetylcholine (ACh) is decreased (Sims et al., 1980) possibly due to a reduction in the uptake of choline via the high affinity choline uptake system (Rylett et al., 1983).
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A number of immunocytochemical studies have indicated the presence of cholinergic neurons in the cerebral cortex of various species of mammals. Whether such cholinergic neurons in the human cerebral cortex are exclusively of subcortical origin is still debated. In this immunocytochemical study, the existence of cortical cholinergic neurons was investigated on surgical samples of human parietal association neocortex using a highly specific monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine biosynthesising enzyme. ChAT immunoreactivity was detected in a subpopulation of neurons located in layers II and III. These were small or medium-sized pyramidal neurons which showed cytoplasmic immunoreactivity in the perikarya and processes, often in close association to blood microvessels. This study, providing demonstration of ChAT neurons in the human parietal neocortex, strongly supports the existence of intrinsic cholinergic innervation of the human neocortex. It is likely that these neurons contribute to the cholinergic innervation of the intracortical microvessels.
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