Title: Acetylcholine release from striatal cholinergic interneurons is controlled differently depending on the firing pattern Arakawa I, Muramatsu I, Uwada J, Sada K, Matsukawa N, Masuoka T Ref: Journal of Neurochemistry, 167:38, 2023 : PubMed
How is the quantal size in neurotransmitter release adjusted for various firing levels? We explored the possible mechanisms that regulate acetylcholine (ACh) release from cholinergic interneurons using an ultra-mini superfusion system. After preloading [(3) H]ACh in rat striatal cholinergic interneurons, the release was elicited by electrical stimulation under a condition in which presynaptic cholinergic and dopaminergic feedback was inhibited. [(3) H]ACh release was reproducible at intervals of more than 10 min; shorter intervals resulted in reduced levels of ACh release. Upon persistent stimulation for 10 min, ACh release transiently increased, before gradually decreasing. Vesamicol, an inhibitor of the vesicular ACh transporter (VAChT), had no effect on the release induced by the first single pulse, but it reduced the release caused by subsequent pulses. Vesamicol also reduced the [(3) H]ACh release evoked by multiple pulses, and the inhibition was enhanced by repetitive stimulation. The decreasing phase of [(3) H]ACh release during persistent stimulation was accelerated by vesamicol treatment. Thus, it is likely that releasable ACh was slowly compensated for via VAChT during and after stimulation, changing the vesicular ACh content. In addition, ACh release per pulse decreased under high-frequency stimulation. The present results suggest that ACh release from striatal cholinergic interneurons may be adjusted by changes in the quantal size due to slow replenishment via VAChT, and by a reduction in release probability upon high-frequency stimulation. These two distinct processes likely enable the fine tuning of neurotransmission and neuroprotection/limitation against excessive output and have important physiological roles in the brain.
        
Title: Focal acetylcholinergic modulation of the human midcingulo-insular network during attention: Meta-analytic neuroimaging and behavioral evidence Chakraborty S, Lee SK, Arnold SM, Haast RAM, Khan AR, Schmitz TW Ref: Journal of Neurochemistry, :, 2023 : PubMed
The basal forebrain cholinergic neurons provide acetylcholine to the cortex via large projections. Recent molecular imaging work in humans indicates that the cortical cholinergic innervation is not uniformly distributed, but rather may disproportionately innervate cortical areas relevant to supervisory attention. In this study, we therefore reexamined the spatial relationship between acetylcholinergic modulation and attention in the human cortex using meta-analytic strategies targeting both pharmacological and non-pharmacological neuroimaging studies. We found that pharmaco-modulation of acetylcholine evoked both increased activity in the anterior cingulate and decreased activity in the opercular and insular cortex. In large independent meta-analyses of non-pharmacological neuroimaging research, we demonstrate that during attentional engagement these cortical areas exhibit (1) task-related co-activation with the basal forebrain, (2) task-related co-activation with one another, and (3) spatial overlap with dense cholinergic innervations originating from the basal forebrain, as estimated by multimodal positron emission tomography and magnetic resonance imaging. Finally, we provide meta-analytic evidence that pharmaco-modulation of acetylcholine also induces a speeding of responses to targets with no apparent tradeoff in accuracy. In sum, we demonstrate in humans that acetylcholinergic modulation of midcingulo-insular hubs of the ventral attention/salience network via basal forebrain afferents may coordinate selection of task relevant information, thereby facilitating cognition and behavior.
        
Title: Whole-brain neural connectivity to cholinergic neurons in the nucleus basalis of Meynert Chen ZY, Yang YL, Li M, Gao L, Qu WM, Huang ZL, Yuan XS Ref: Journal of Neurochemistry, 166:233, 2023 : PubMed
The cholinergic neurons in the nucleus basalis of Meynert (NBM) are a key structure in cognition, the dysfunction of which is associated with various neurological disorders, especially dementias. However, the whole-brain neural connectivity to cholinergic neurons in the NBM remains to be further and comprehensively researched. Using virus-based, specific, retrograde, and anterograde tracing, we illustrated the monosynaptic inputs and axon projections of NBM cholinergic neurons in choline acetyltransferase (ChAT)-Cre transgenic mice. Our results showed that NBM cholinergic neurons received mainly inputs from the caudate putamen and the posterior limb of the anterior commissure in the subcortex. Moreover, the majority of cholinergic terminals from the NBM were observed in the cortex mantle, including the motor cortex, sensory cortex, and visual cortex. Interestingly, although NBM cholinergic neurons received input projections from the caudate putamen, interstitial nucleus of the posterior limb of the anterior commissure, and central amygdaloid nucleus, NBM cholinergic neurons sparsely sent axon projection to innervate these areas. Furthermore, primary motor cortex, secondary motor cortex, and primary somatosensory cortex received abundant inputs from the NBM but sent few outputs to the NBM. Taken together, our results reveal the detailed and specific connectivity of cholinergic neurons of the NBM and provide a neuroanatomic foundation for further studies to explore the important physiological functions of NBM cholinergic neurons.
        
Title: Butyrylcholinesterase in lipid metabolism: A new outlook Gok M, Cicek C, Bodur E Ref: Journal of Neurochemistry, :, 2023 : PubMed
Cholinesterase enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are traditionally associated with the termination of acetylcholine mediated neural signaling. The fact that these ubiquitous enzymes are also found in tissues not involved in neurotransmission has led to search for alternative functions for these enzymes. Cholinesterases are reported to be involved in many lipid related disease states. Taking into view that lipases and cholinesterases belong to the same enzyme class and by comparing the catalytic sites, we propose a new outlook on the link between BChE and lipid metabolism. The lipogenic substrates of BChE that have recently emerged in contrast to traditional cholinesterase substrates are explained through the hydrolytic capacity of BChE for ghrelin, 4-methyumbelliferyl (4-mu) palmitate, and arachidonoylcholine and through endogenous lipid mediators such as cannabinoids like anandamide and essential fatty acids. The abundance of BChE in brain, intestine, liver, and plasma, tissues with active lipid metabolism, supports the idea that BChE may be involved in lipid hydrolysis. BChE is also regulated by various lipids such as linoleic acid, alpha-linolenic acid or dioctanoylglycerol, whereas AChE is inhibited. The finding that BChE is able to hydrolyze 4-mu palmitate at a pH where lipases are less efficient points to its role as a backup in lipolysis. In diseases such as Alzheimer, in which elevated BChE and impaired lipid levels are observed, the lipolytic activity of BChE might be involved. It is possible to suggest that fatty acids such as 4-mu palmitate, ghrelin, arachidonoylcholine, essential fatty acids, and other related lipid mediators regulate cholinesterases, which could lead to some sort of compensatory mechanism at high lipid concentrations.
        
Title: Recent advances in cholinergic mechanisms as reactions to toxicity, stress, and neuroimmune insults Kovarik Z, Moshitzky G, Macek Hrvat N, Soreq H Ref: Journal of Neurochemistry, :, 2023 : PubMed
This review presents recent studies of the chemical and molecular regulators of acetylcholine (ACh) signaling and the complexity of the small molecule and RNA regulators of those mechanisms that control cholinergic functioning in health and disease. The underlying structural, neurochemical, and transcriptomic concepts, including basic and translational research and clinical studies, shed new light on how these processes inter-change under acute states, age, sex, and COVID-19 infection; all of which modulate ACh-mediated processes and inflammation in women and men and under diverse stresses. The aspect of organophosphorus (OP) compound toxicity is discussed based on the view that despite numerous studies, acetylcholinesterase (AChE) is still a vulnerable target in OP poisoning because of a lack of efficient treatment and the limitations of oxime-assisted reactivation of inhibited AChE. The over-arching purpose of this review is thus to discuss mechanisms of cholinergic signaling dysfunction caused by OP pesticides, OP nerve agents, and anti-cholinergic medications; and to highlight new therapeutic strategies to combat both the acute and chronic effects of these chemicals on the cholinergic and neuroimmune systems. Furthermore, OP toxicity was examined in view of cholinesterase inhibition and beyond in order to highlight improved small molecules and RNA therapeutic strategies and assess their predicted pitfalls to reverse the acute toxicity and long-term deleterious effects of OPs.
        
Title: Recent advances in cholinergic mechanisms: A preface for the ISCM2022 special issue Kovarik Z, Soreq H Ref: Journal of Neurochemistry, :, 2023 : PubMed
This preface introduces the Journal of Neurochemistry special issue on Cholinergic Mechanisms that highlights the progress in the molecular, structural, neurochemical, pharmacological, toxicological, and clinical studies of the cholinergic system which underline its complexity and impact on health and disease. This issue comprises of (systematic) reviews and original articles, the majority of which have been presented at the 17th International Symposium on Cholinergic Mechanisms (ISCM2022) held in Dubrovnik, Croatia in May 2022. The symposium brought together leading "Cholinergikers" to shed new light on cholinergic transmission, ranging from the molecular to the clinical and cognitive mechanisms.
        
Title: How can I measure brain acetylcholine levels in vivo? Advantages and caveats of commonly used approaches Mineur YS, Picciotto MR Ref: Journal of Neurochemistry, 167:3, 2023 : PubMed
The neurotransmitter acetylcholine (ACh) plays a central role in the regulation of multiple cognitive and behavioral processes, including attention, learning, memory, motivation, anxiety, mood, appetite, and reward. As a result, understanding ACh dynamics in the brain is essential for elucidating the neural mechanisms underlying these processes. In vivo measurements of ACh in the brain have been challenging because of the low concentrations and rapid turnover of this neurotransmitter. Here, we review a number of techniques that have been developed to measure ACh levels in the brain in vivo. We follow this with a deeper focus on use of genetically encoded fluorescent sensors coupled with fiber photometry, an accessible technique that can be used to monitor neurotransmitter release with high temporal resolution and specificity. We conclude with a discussion of methods for analyzing fiber photometry data and their respective advantages and disadvantages. The development of genetically encoded fluorescent ACh sensors is revolutionizing the field of cholinergic signaling, allowing temporally precise measurement of ACh release in awake, behaving animals. Use of these sensors has already begun to contribute to a mechanistic understanding of cholinergic modulation of complex behaviors.
        
Title: Connectivity between surface and interior in catalytic subunits of acetylcholinesterases inferred from their X-ray structures Radic Z Ref: Journal of Neurochemistry, :, 2023 : PubMed
Catalytic activity and function of acetylcholinesterase (AChE; EC 3.1.1.7) have been recognized and studied for over a century, its quaternary and primary structures for about half a century and its tertiary structure has been known for about 33 years. Clear understanding of relationships between the structure and the function is still pending for this enzyme. Hundreds of crystallographic, static snapshots of AChEs from different sources reveal largely one general backbone conformation with narrow entry into the active center gorge, tightly fit to accept one acetylcholine (ACh) molecule, in contrast to its high catalytic turnover. This short review of available X-ray structures of AChEs from electric ray Torpedo californica, mouse and human, finds some limited, yet consistent deviations in conformations of selected secondary structure elements of AChE relevant for its function. The observed conformational diversity of the acyl pocket loop of AChE, unlike the large -loop, appears consistent with structurally dynamic INS data and solution-based SAXS experiments to explain its dominant role in controlling the size of the active center gorge opening, as well as connectivity between the immediate surroundings of the buried active Ser, and catalytically relevant sites on the AChE surface.
        
Title: Detection of early Alzheimer's disease-like molecular alterations in a mouse model expressing human ApoE4 Rai A, Ojiakor OA, Rylett RJ Ref: Journal of Neurochemistry, 166:572, 2023 : PubMed
The E4 allele of apolipoprotein E (ApoE4) is a key genetic risk factor for late-onset Alzheimer's disease (AD), increasing the risk of developing the disease by up to three-fold. However, the mechanisms by which ApoE4 contributes to AD pathogenesis are poorly understood. Here, we utilize a mouse model expressing either human ApoE3 or human ApoE4 to examine the effects of the E4 allele on a wide range of genetic and molecular pathways that are altered in early AD pathology. We demonstrate that ApoE4-expressing mice begin to show early differential expression of multiple genes, leading to alterations in downstream pathways related to neural cell maintenance, insulin signaling, amyloid processing and clearance, and synaptic plasticity. These alterations may result in the earlier accumulation of pathological proteins such as beta-amyloid that may build up within cells, leading to the accelerated degeneration of neurons and astrocytes as observed in ApoE4-positive individuals. We also examine the metabolic effects associated with a high-fat diet (HFD) in male ApoE4-expressing mice compared with regular chow diet (RD) fed mice at different ages. We found that young ApoE4-expressing mice fed HFD developed metabolic disturbances, such as elevated weight gain, blood glucose, and plasma insulin levels that cumulatively have been observed to increase the risk of AD in humans. Taken together, our results reveal early pathways that could mediate ApoE4-related AD risk and may help identify more tractable therapeutic targets for treating ApoE4-associated AD.
Millions of individuals globally suffer from inadvertent, occupational or self-harm exposures from organophosphate (OP) insecticide exposures; significantly impacting human health. Similar to nerve agents, insecticides are neurotoxins which target and inhibit acetylcholinesterase (AChE) in central and peripheral synapses in the cholinergic nervous system. Post-exposure therapeutic countermeasures generally include admin-istration of atropine with an oxime, to reactivate the OP-inhibited AChE. However, animal model studies and recent clinical trials using insecticide-poisoned individuals have shown minimal clinical benefits of the currently approved oximes and their efficacy as antidotes has been debated. Currently-used oximes either reactivate poorly, do not readily cross the blood brain barrier (BBB), or are rapidly cleared from the circulation and must be repeatedly administered. Zwitterionic oximes of unbranched and simplified structure eg RS194B have been developed that efficiently cross the BBB resulting in reactivation of OP-inhibited AChE and dramatic reversal of severe clinical symptoms in mice and macaques exposed to OP insecticide or nerve agents. Thus, a single IM injection of RS194B has been shown to rapidly restore blood AChE and butyrylcholinesterase (BChE) activity, reverse cholinergic symptoms and prevent death in macaques following lethal inhaled sarin and paraoxon exposure. The present macaque studies extend these studies and assess the ability of post-exposure RS194B treatment to counteract oral poisoning by highly toxic diethylphosphorothioate insecticides such as parathion and chlorpyrifos, which require oxidation by P450 in the liver to convert inactive thions to the active toxic oxon forms, and once again demonstrated its efficacy to reactivate and alleviate clinical symptoms within 60 mins of a single IM administration. Furthermore, when delivered orally, the Tmax of RS194B at 1-2 hours was in the same range as those administered IM but were maintained in the circulation for longer periods greatly facilitating the use of RS194B as a non-invasive treatment, especially in isolated rural settings.
People bitten by Alpine vipers are usually treated with antivenom antisera to prevent the noxious consequences caused by the injected venom. However, this treatment suffers from a number of drawbacks and additional therapies are necessary. The venoms of Vipera ammodytes and of Vipera aspis are neurotoxic and cause muscle paralysis by inducing neurodegeneration of motor axon terminals because they contain a presynaptic acting sPLA(2) neurotoxin. We have recently found that any type of damage to motor axons is followed by the expression and activation of the intercellular signaling axis consisting of the CXCR4 receptor present on the membrane of the axon stump and of its ligand, the chemokine CXCL12 released by activated terminal Schwann cells. We show here that also V. ammodytes and V. aspis venoms cause the expression of the CXCL12-CXCR4 axis. We also show that a small molecule agonist of CXCR4, dubbed NUCC-390, induces a rapid regeneration of the motor axon terminal with functional recovery of the neuromuscular junction. These findings qualify NUCC-390 as a promising novel therapeutics capable of improving the recovery from the paralysis caused by the snakebite of the two neurotoxic Alpine vipers.
The alpha7 nicotinic receptors (NR) have been confirmed in the heart but their role in cardiac functions has been contradictory. To address these contradictory findings, we analyzed cardiac functions in alpha7 NR knockout mice (alpha7(-/-) ) in vivo and ex vivo in isolated hearts. A standard limb leads electrocardiogram was used, and the pressure curves were recorded in vivo, in Arteria carotis and in the left ventricle, or ex vivo, in the left ventricle of the spontaneously beating isolated hearts perfused following Langedorff's method. Experiments were performed under basic conditions, hypercholinergic conditions, and adrenergic stress. The relative expression levels of alpha and beta NR subunits, muscarinic receptors, beta1 adrenergic receptors, and acetylcholine life cycle markers were determined using RT-qPCR. Our results revealed a prolonged QT interval in alpha7(-/-) mice. All in vivo hemodynamic parameters were preserved under all studied conditions. The only difference in ex vivo heart rate between genotypes was the loss of bradycardia in prolonged incubation of isoproterenol-pretreated hearts with high doses of acetylcholine. In contrast, left ventricular systolic pressure was lower under basal conditions and showed a significantly higher increase during adrenergic stimulation. No changes in mRNA expression were observed. In conclusion, alpha7 NR has no major effect on heart rate, except when stressed hearts are exposed to a prolonged hypercholinergic state, suggesting a role in acetylcholine spillover control. In the absence of extracardiac regulatory mechanisms, left ventricular systolic impairment is revealed.