Exploring the Regulation of the Expression of ChAT and VAChT Genes in NG108-15 Cells: Implication of PKA and PI3K Signaling Pathways

Involvement of different protein kinases regulated by cAMP and implication of muscarinic receptors in the regulation of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) mRNA levels and ChAT activity has been studied in NG108-15 cells. Dibutyryl cAMP enhanced both ChAT and VAChT mRNA levels and stimulated ChAT activity. Muscarinic stimulation or inhibition did not change ChAT activity or the receptor subtype mRNA pattern. MEK1/2 did not affect the regulation of ChAT and VAChT mRNA levels. However, PKA plays a major role in regulating ChAT and VAChT mRNA levels, because H89 decreased both. Strikingly, inhibition of PI3K by LY294002 had two opposite effects: ChAT mRNA level was decreased and VAChT mRNA level was increased. Such a result consolidates the observation that ChAT and VAChT genes, despite their unusual organization in a single cholinergic locus, can be differentially or synergistically regulated, depending on the activated signaling pathways.     

Cholinergic deficit in Alzheimer's disease: A study based on CSF and autopsy data

Cholinesterase (ChE) activity was measured as a possible marker of cholinergic neurotransmission of the brain in CSF of 93 patients with probable Alzheimer's disease/senile dementia of the Alzheimer type (AD/SDAT) and of 29 control patients. ChE activity in CSF was decreased significantly in the AD/SDAT patients as compared to the controls. This reduction correlated significantly with the various measures of the severity of dementia. However, the reduction of ChE activity was only moderate (25–30%) even in patients with the most severe dementia and nonsignificant in patients with early symptoms of AD/SDAT. The significance of various confounding factors, which may interfere with CSF ChE measurements is discussed. Our findings seem to indicate that the deficiency of cholinergic neurons is not directly reflected in CSF and that the measurements of ChE activities in CSF are not helpful in diagnosing AD/SDAT. In the autopsy study the activities of cholineacetyltransferase (ChAT) and ChE were determined for ten brain areas of 20 AD/SDAT patients and of 14 controls. In AD/SDAT patients ChAT activity was profoundly decreased (50–85% decrease) in the cortical areas and hippocampus, but was unchanged or only mildly reduced in other subcortical brain areas. This study further confirms that the affection of cholinergic neurons is limited to projections from nucleus basalis to cortex and hippocampus, whereas other cholinergic neurons, like in striatum, seem to be relatively spared. In general, the activities of ChAT and ChE were lower in Alzheimer patients dying at younger age suggesting more severe disease process with these patients.     

Choline acetyltransferase activity at different ages in brain of Ts65Dn mice, an animal model for Down's syndrome and related neurodegenerative diseases

Ts65Dn mice, trisomic for a portion of chromosome 16 segmentally homologous to human chromosome 21, are an animal model for Down's syndrome and related neurodegenerative diseases, such as dementia of the Alzheimer type. In these mice, cognitive deficits and alterations in number of basal forebrain cholinergic neurons have been described. We have measured in Ts65Dn mice the catalytic activity of the cholinergic marker, choline acetyltransferase (ChAT), as well as the activity of the acetylcholine-degrading enzyme acetylcholinesterase (AChE), in the hippocampus and in cortical targets of basal forebrain cholinergic neurons. In mice aged 10 months, ChAT activity was significantly higher in Ts65Dn mice, compared to 2N animals, in the hippocampus, olfactory bulb, olfactory cortex, pre-frontal cortex, but not in other neocortical regions. At 19 months of age, on the other hand, no differences in ChAT activity were found. Thus, alterations of ChAT activity in these forebrain areas seem to recapitulate those recently described in patients scored as cases of mild cognitive impairment or mild Alzheimer's disease. Other neurochemical markers putatively associated with the disease progression, such as those implicating astrocytic hyperactivity and overproduction of amyloid precursor protein family, were preferentially found altered in some brain regions at the oldest age examined (19 months).     

Transcriptomic effects of depleted uranium on acetylcholine and cholesterol metabolisms in Alzheimer's disease model

Some heavy metals, or aluminium, could participate in the development of Alzheimer disease (AD). Depleted uranium (DU), another heavy metal, modulates the cholinergic system and the cholesterol metabolism in the brain of rats, but without neurological disorders. The aim of this study was to determine what happens in organisms exposed to DU that will/are developing the AD. This study was thus performed on a transgenic mouse model for human amyloid precursor protein (APP), the Tg2576 strain. The possible effects of DU through drinking water (20 mg/L) over an 8-month period were analyzed on acetylcholine and cholesterol metabolisms at gene level in the cerebral cortex. The mRNA levels of choline acetyl transferase (ChAT) vesicular acetylcholine transporter (VAChT) and ATP-binding cassette transporter A1 (ABC A1) decreased in control Tg2576 mice in comparison with wild-type mice (respectively -89%, -86% and -44%, p < 0.05). Chronic exposure of Tg2576 mice to DU increased mRNA levels of ChAT (+189%, p < 0.05), VAChT (+120%, p < 0.05) and ABC A1 (+52%, p < 0.05) compared to control Tg2576 mice. Overall, these modifications of acetylcholine and cholesterol metabolisms did not lead to increased disturbances that are specific of AD, suggesting that chronic DU exposure did not worsen the pathology in this experimental model.     

Functional Regulation of Choline Acetyltransferase by Phosphorylation

Choline acetyltransferase (ChAT) catalyzes synthesis of acetylcholine (ACh) in cholinergic neurons. ACh synthesis is regulated by availability of precursors choline and acetyl coenzyme A or by activity of ChAT; ChAT regulates ACh synthesis under some conditions. Posttranslational phosphorylation is a common mechanism for regulating the function of proteins. Analysis of the primary sequence of 69-kD human ChAT indicates that it has putative phosphorylation consensus sequences for multiple protein kinases. ChAT is phosphorylated on serine-440 and threonine-456 by protein kinase C and CaM kinase II, respectively. These phosphorylation events regulate activity of the enzyme, as well as its binding to plasma membrane and interaction with other cellular proteins. It is relevant to investigate differences in constitutive and inducible patterns of phosphorylation of ChAT under physiological conditions and in response to challenges that cholinergic neurons may be exposed to, and to determine how changes in phosphorylation relate to changes in neurochemical transmission.     

Influence of retinoic acid and of cyclic AMP on the expression of choline acetyltransferase and of vesicular acetylcholine transporter in NG108-15 cells

Treatment of the cholinergic cell line NG108-15 with retinoic acid or cAMP results in an increase of choline acetyltransferase activity (ChAT) whereas none of these agents influences the amount of the vesicular acetylcholine transporter (VAChT) as judged from vesamicol binding and immunoblot studies. We suggest that immaturity of posttranslational events controlling the expression of VAChT protein is responsible for the apparent absence of coregulation of ChAT and VAChT protein expression.     

Immunohistochemical characterisation of cholinergic neurons in the anterior pelvic ganglion of the male pig

This study investigated immunohistochemical properties of cholinergic neurons in the anterior pelvic ganglion (APG) of juvenile male pigs (n=7). Cholinergic neurons were identified using antibodies against choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT). Immunoblotting was applied to verify the specificity of ChAT-immunostaining. Western blotting performed on APG tissue homogenates detected single immunoreactive protein with a molecular weight matching that of ChAT (71.6 kDa). It was found that many APG neurons expressed immunoreactivity to ChAT or VAChT (40% and 39% of the neurons, respectively). The analysis of adjacent sections from the ganglion revealed complete colocalization of ChAT and VAChT in these nerve cells. Furthermore, virtually all the ChAT-positive neurons were tyrosine hydroxylase (TH)-negative (non-adrenergic) but many of them displayed immunoreactivity to nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) or somatostatin (SOM). There were also single nerve cell bodies that stained for neither ChAT nor TH. The comparison of the adjacent sections revealed that NOS, VIP, NPY and SOM were simultaneously co-expressed in the majority of the cholinergic somata. ChAT- or VAChT-positive varicose nerve terminals supplied nearly all neuronal profiles within the ganglion often forming loose basket-like formations surrounding the particular nerve cell bodies. The present study for the first time has revealed that nearly all non-adrenergic neurons in the porcine APG are cholinergic in nature, i.e. express immunoreactivity for ChAT and VAChT. Considering a high coincidence between the chemical coding of non-adrenergic (cholinergic) nerve fibres supplying some porcine male reproductive organs described in earlier papers and that of cholinergic pelvic neurons found in this study it is further concluded that pelvic ganglia are probably the major source of cholinergic innervation for the porcine urogenital system.     

Oral Administration of Gintonin Attenuates Cholinergic Impairments by Scopolamine,Amyloid-β Protein,and Mouse Model of Alzheimer’s Disease

Gintonin is a novel ginseng-derived lysophosphatidic acid (LPA) receptor ligand. Oral administration of gintonin ameliorates learning and memory dysfunctions in Alzheimer’s disease (AD) animal models. The brain cholinergic system plays a key role in cognitive functions. The brains of AD patients show a reduction in acetylcholine concentration caused by cholinergic system impairments. However, little is known about the role of LPA in the cholinergic system. In this study, we used gintonin to investigate the effect of LPA receptor activation on the cholinergic system in vitro and in vivo using wild-type and AD animal models. Gintonin induced [Ca²⁺]_i transient in cultured mouse hippocampal neural progenitor cells (NPCs). Gintonin-mediated [Ca²⁺]_i transients were linked to stimulation of acetylcholine release through LPA receptor activation. Oral administration of gintonin-enriched fraction (25, 50, or 100 mg/kg, 3 weeks) significantly attenuated scopolamine-induced memory impairment. Oral administration of gintonin (25 or 50 mg/kg, 2 weeks) also significantly attenuated amyloid-β protein (Aβ)-induced cholinergic dysfunctions, such as decreased acetylcholine concentration, decreased choline acetyltransferase (ChAT) activity and immunoreactivity, and increased acetylcholine esterase (AChE) activity. In a transgenic AD mouse model, long-term oral administration of gintonin (25 or 50 mg/kg, 3 months) also attenuated AD-related cholinergic impairments. In this study, we showed that activation of G protein-coupled LPA receptors by gintonin is coupled to the regulation of cholinergic functions. Furthermore, this study showed that gintonin could be a novel agent for the restoration of cholinergic system damages due to Aβ and could be utilized for AD prevention or therapy.     

VAChT-Cre. Fast and VAChT-Cre.Slow: postnatal expression of Cre recombinase in somatomotor neurons with different onset

The cholinergic gene locus (CGL) consists of the genes encoding the choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT). To establish a cholinergic-specific Cre-expressing mouse, we constructed a transgene expression vector (VAChT-Cre) with 11.3 kb human CGL in which a Cre-IRES-EGFP unit was inserted in the VAChT open reading frame. The activity of Cre, whose expression was driven by the VAChT promoter, was examined by crossing a reporter mouse (CAG-CAT-Z) in which expression of LacZ is activated upon Cre-mediated recombination. Transgenic lines with the VAChT-Cre construct displayed the restricted Cre expression in a subset of cholinergic neurons in the somatomotor nuclei and medial habenular nucleus, but absent in visceromotor and other central and peripheral cholinergic neurons. Cre expression was first observed at postnatal day 7 and later detected in approximately 40-60% of somatomotor neurons. Based on the onset of Cre expression, we generated two mouse lines (two alleles; VAChT-Cre. Fast and VAChT-Cre.Slow) in which Cre expression reaches maximal levels fast and slow, respectively. The use of VAChT-Cre mice should allow us to deliver Cre to a subset of postnatal motor neurons, thereby bypassing lethality and facilitating analysis of gene function in adult motor neurons.     

Coordinate Expression of the Vesicular Acetylcholine Transporter and Choline Acetyltransferase Following Septohippocampal Pathway Lesions

Abstract: The gene for the vesicular acetylcholine transporter (VAChT) was recently cloned and found to be located within a 5' noncoding intron of the gene for choline acetyltransferase (ChAT). There appear to be several shared and unique promoters for each gene, suggesting that control of expression of these two genes can be either coordinated or independent. Two lesions, axotomy and immunotoxin, directed at the well defined septohippocampal cholinergic pathway were used to determine VAChT and ChAT protein expression in the degenerating terminal fields in the hippocampus and the cell bodies of the medial septum nucleus after injury. Two weeks after lesioning, decreases of up to 90% in VAChT were found in the affected hippocampus by immunoblotting and immunocytochemistry, similar to ChAT activity. The number of VAChT- and ChAT-immunopositive neurons in the medial septum decreased by up to 95%. Eight weeks following axotomy, the number of VAChT- and ChAT-immunopositive neurons had increased to almost 50% in fimbria-fornix-lesioned animals, indicating coordinate reexpression of both cholinergic markers in recovered neurons. There was no recovery of either VAChT or ChAT immunoreactivity after the irreversible immunotoxin lesions. Thus, with use of immunological techniques, there appears to be coordinate expression of VAChT and ChAT in the septohippocampal pathway following either unilateral fimbria-fornix or bilateral immunotoxin lesion.     

Cytosolic Choline Acetyltransferase Binds Specifically to Cholinergic Plasma Membrane of Rat Brain Synaptosomes to Generate Membrane-Bound Enzyme

Uncovering the way membrane-bound choline acetyltransferase (ChAT) interacts with membranes and with which membrane in cholinergic neurons may help in understanding its role in acetylcholine metabolism. Subfractionation of rat hippocampal synaptosomes aiming to separate synaptic vesicles from plasma membranes shows that membrane-bound ChAT is bound to plasma membrane. Either detergents or urea and alkali can solubilize membrane-bound enzyme. Detergent-solubilized enzyme has a higher sedimentation rate than urea-alkali solubilized or cytosolic ChAT. Once dissociated, membrane-bound ChAT reassociates specifically with cholinergic plasma membranes, a process that was abolished by previous treatment of membranes with trypsin. Cytosolic ChAT behaves similarly. Thus, in cholinergic synaptosomes, ChAT exists as cytosolic and peripheral activity. Cytosolic ChAT generates peripheral enzyme most probably by interacting with a protein of plasma membrane of cholinergic nerve terminals. This receptor protein might regulate the amount of membrane-bound ChAT in cholinergic neurons.     

Single dose exposure of sarin and physostigmine differentially regulates expression of choline acetyltransferase and vesicular acetylcholine transporter in rat brain

Choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) are the key components of cholinergic system apart from acetylcholinesterase. Effects of subcutaneous exposures of 0.25 and 0.5 LD(50) sarin and 0.75 mg/kg physostigmine on immunoreactivity levels of these two proteins (ChAT and VAChT) were studied. Immunoreactivity levels of ChAT decreased significantly after 1 and 3 days in cortex and 3 days of 0.25 LD(50) sarin administration in cerebellum. While 0.5 LD(50) sarin exposure caused significant down regulation after 2.5 h to 7 days in cortex and 1 and 3 days in cerebellum with respect to controls. Physostigmine at 0.75 mg/kg dose showed enhanced levels of ChAT after 1 day which decreased significantly after 3 and 7 days both in cortex and cerebellum compared to controls. VAChT level decreased significantly after 1 day in cortex and 3 and 7 days in cerebellum after 0.25 LD(50) sarin administration, while 0.5 LD(50) sarin significantly lowered VAChT immunoreactivity level after 2.5 h and 7 days in cortex and 2.5 h and 1 day in cerebellum. Physostigmine at 0.75 mg/kg dose showed significant enhanced immunoreactivity levels of VAChT after 1, 3, and 7 days in cortex and 3 days in cerebellum. Results show that acetylcholinesterase inhibition by sarin caused reduction in cholinergic neurotransmission at cholinergic proteins expression levels, while physostigmine caused differential expression of key cholinergic proteins. Moreover, cortex, which receives greater cholinergic innervations, is more susceptible to anticholinesterase effect on cholinergic gene expression. These changes can explain delayed neurocognitive changes during anticholinesterases induced chronic neurotoxicity.     

Activation of GSK‐3 disrupts cholinergic homoeostasis in nucleus basalis of Meynert and frontal cortex of rats

The cholinergic impairment is an early marker in Alzheimer's disease (AD), while the mechanisms are not fully understood. We investigated here the effects of glycogen synthase kinse‐3 (GSK‐3) activation on the cholinergic homoeostasis in nucleus basalis of Meynert (NBM) and frontal cortex, the cholinergic enriched regions. We activated GSK‐3 by lateral ventricular infusion of wortmannin (WT) and GF‐109203X (GFX), the inhibitors of phosphoinositol‐3 kinase (PI3‐K) and protein kinase C (PKC), respectively, and significantly decreased the acetylcholine (ACh) level via inhibiting choline acetyl transferase (ChAT) rather than regulating acetylcholinesterase (AChE). Neuronal axonal transport was disrupted and ChAT accumulation occurred in NBM and frontal cortex accompanied with hyperphosphorylation of tau and neurofilaments. Moreover, ChAT expression decreased in NBM attributing to cleavage of nuclear factor‐κB/p100 into p52 for translocation into nucleus to lower ChAT mRNA level. The cholinergic dysfunction could be mimicked by overexpression of GSK‐3 and rescued by simultaneous administration of LiCl or SB216763, inhibitors of GSK‐3. Our data reveal the molecular mechanism that may underlie the cholinergic impairments in AD patients.     

Newly raised anti‐VAChT and anti‐ChAT antibodies detect cholinergic cells in chicken embryos

The autonomic nervous system consists of sympathetic and parasympathetic nerves, which functionally antagonize each other to control physiology and homeostasis of organs. However, it is largely unexplored how the autonomic nervous system is established during development. In particular, early formation of parasympathetic network remains elusive because of its complex anatomical structure. To distinguish between parasympathetic (cholinergic) and sympathetic (adrenergic) ganglia, vesicular acetylcholine transporter (VAChT) and choline O‐acetyltransferase (ChAT), proteins associated with acetylcholine synthesis, are known to be useful markers. Whereas commercially available antibodies against these proteins are widely used for mammalian specimens including mice and rats, these antibodies do not work satisfactorily in chickens, although chicken is an excellent model for the study of autonomic nervous system. Here, we newly raised antibodies against chicken VAChT and ChAT proteins. One monoclonal and three polyclonal antibodies for VAChT, and one polyclonal antibody for ChAT were obtained, which were available for Western blotting analyses and immunohistochemistry. Using these verified antibodies, we detected cholinergic cells in Remak ganglia of autonomic nervous system, which form in the dorsal aspect of the digestive tract of chicken E13 embryos. The antibodies obtained in this study are useful for visualization of cholinergic neurons including parasympathetic ganglia.