Frontal Cortex Lipid Alterations During the Onset of Alzheimer's Disease.

Background: Although sporadic Alzheimer's disease (AD) is a neurodegenerative disorder of unknown etiology, familial AD is associated with specific gene mutations. A commonality between these forms of AD is that both display multiple pathogenic events including cholinergic and lipid dysregulation. Objective: We aimed to identify the relevant lipids and the activity of their related receptors in the frontal cortex and correlating them with cognition during the progression of AD. Methods: MALDI-mass spectrometry imaging (MSI) and functional autoradiography was used to evaluate the distribution of phospholipids/sphingolipids and the activity of cannabinoid 1 (CB1), sphingosine 1-phosphate 1 (S1P1), and muscarinic M2/M4 receptors in the frontal cortex (FC) of people that come to autopsy with premortem clinical diagnosis of AD, mild cognitive impairment (MCI), and no cognitive impairment (NCI). Results: MALDI-MSI revealed an increase in myelin-related lipids, such as diacylglycerol (DG) 36:1, DG 38:5, and phosphatidic acid (PA) 40:6 in the white matter (WM) in MCI compared to NCI, and a downregulation of WM phosphatidylinositol (PI) 38:4 and PI 38:5 levels in AD compared to NCI. Elevated levels of phosphatidylcholine (PC) 32:1, PC 34:0, and sphingomyelin 38:1 were observed in discrete lipid accumulations in the FC supragranular layers during disease progression. Muscarinic M2/M4 receptor activation in layers V-VI decreased in AD compared to MCI. CB1 receptor activity was upregulated in layers V-VI, while S1P1 was downregulated within WM in AD relative to NCI. Conclusions: FC WM lipidomic alterations are associated with myelin dyshomeostasis in prodromal AD, suggesting WM lipid maintenance as a potential therapeutic target for dementia.

Neuroprotection of Cholinergic Neurons with a Tau Aggregation Inhibitor and Rivastigmine in an Alzheimer's-like Tauopathy Mouse Model.

Basal forebrain cholinergic dysfunction, most likely linked with tau protein aggregation, is a characteristic feature of Alzheimer's disease (AD). Recent evidence suggests that tau protein is a putative target for the treatment of dementia, and the tau aggregation inhibitor, hydromethylthionine mesylate (HMTM), has emerged as a potential disease-modifying treatment. However, its efficacy was diminished in patients already receiving approved acetylcholinesterase inhibitors. In this study, we ask whether this negative interaction can also be mimicked in experimental tau models of AD and whether the underlying mechanism can be understood. From a previous age profiling study, 6-month-old line 1 (L1) tau transgenic mice were characterized by a severe reduction in several cholinergic markers. We therefore assessed whether long-term pre-exposure with the acetylcholinesterase inhibitor rivastigmine alone and in conjunction with the tau aggregation inhibitor HMTM can reverse cholinergic deficits in L1. Rivastigmine and HMTM, and combinations of the two compounds were administered orally for 11 weeks to both L1 and wild-type mice. The brains were sectioned with a focus on the basal forebrain, motor cortex and hippocampus. Immunohistochemical staining and quantification of choline acetyltransferase (ChAT), tyrosine kinase A (TrkA)-positive neurons and relative optical intensity (ROI) for vesicular acetylcholine transporter (VAChT), and acetylcholinesterase (AChE) reactivity confirmed reversal of the diminished cholinergic phenotype of interneurons (nucleus accumbens, striatum) and projection neurons (medial septum, nucleus basalis magnocellularis) by HMTM, to a greater extent than by rivastigmine alone in L1 mice. Combined administration did not yield additivity but, in most proxies, led to antagonistic effects in which rivastigmine decreased the benefits shown with HMTM alone. Local markers (VAChT and AChE) in target structures of the basal forebrain, motor cortex and hippocampal CA3 seemed to be normalized by HMTM, but not by rivastigmine or the combination of both drugs. HMTM, which was developed as a tau aggregation inhibitor, strongly decreased the tau load in L1 mice, however, not in combination with rivastigmine. Taken together, these data confirm a cholinergic phenotype in L1 tau transgenic mice that resembles the deficits observed in AD patients. This phenotype is reversible by HMTM, but at the same time appears to be subject to a homeostatic regulation induced by chronic pre-treatment with an acetylcholinesterase inhibitor, which interferes with the efficacy of HMTM. The strongest phenotypic reversal coincided with a normalization of the tau load in the cortex and hippocampus of L1, suggesting that tau accumulation underpins the loss of cholinergic markers in the basal forebrain and its projection targets.

The following article for this Special Issue was previously published and can be found in its respective issue online: "Acetylcholine release from striatal cholinergic interneurons is controlled differently depending on the firing pattern".

Acetylcholine release from striatal cholinergic interneurons is controlled differently depending on the firing pattern (Published in JNC 167.1 issue) https://onlinelibrary.wiley.com/doi/10.1111/jnc.15950.

Postsynaptic receptors regulate presynaptic transmitter stability through transsynaptic bridges.

Stable matching of neurotransmitters with their receptors is fundamental to synapse function and reliable communication in neural circuits. Presynaptic neurotransmitters regulate the stabilization of postsynaptic transmitter receptors. Whether postsynaptic receptors regulate stabilization of presynaptic transmitters has received less attention. Here, we show that blockade of endogenous postsynaptic acetylcholine receptors (AChR) at the neuromuscular junction destabilizes the cholinergic phenotype in motor neurons and stabilizes an earlier, developmentally transient glutamatergic phenotype. Further, expression of exogenous postsynaptic gamma-aminobutyric acid type A receptors (GABAA receptors) in muscle cells stabilizes an earlier, developmentally transient GABAergic motor neuron phenotype. Both AChR and GABAA receptors are linked to presynaptic neurons through transsynaptic bridges. Knockdown of specific components of these transsynaptic bridges prevents stabilization of the cholinergic or GABAergic phenotypes. Bidirectional communication can enforce a match between transmitter and receptor and ensure the fidelity of synaptic transmission. Our findings suggest a potential role of dysfunctional transmitter receptors in neurological disorders that involve the loss of the presynaptic transmitter.

Functional neuroanatomy of basal forebrain projections to the basolateral amygdala: Transmitters, receptors, and neuronal subpopulations.

The projections of the basal forebrain (BF) to the hippocampus and neocortex have been extensively studied and shown to be important for higher cognitive functions, including attention, learning, and memory. Much less is known about the BF projections to the basolateral nuclear complex of the amygdala (BNC), although the cholinergic innervation of this region by the BF is actually far more robust than that of cortical areas. This review will focus on light and electron microscopic tract-tracing and immunohistochemical (IHC) studies, many of which were published in the last decade, that have analyzed the relationship of BF inputs and their receptors to specific neuronal subtypes in the BNC in order to better understand the anatomical substrates of BF-BNC circuitry. The results indicate that BF inputs to the BNC mainly target the basolateral nucleus of the BNC (BL) and arise from cholinergic, GABAergic, and perhaps glutamatergic BF neurons. Cholinergic inputs mainly target dendrites and spines of pyramidal neurons (PNs) that express muscarinic receptors (MRs). MRs are also expressed by cholinergic axons, as well as cortical and thalamic axons that synapse with PN dendrites and spines. BF GABAergic axons to the BL also express MRs and mainly target BL interneurons that contain parvalbumin. It is suggested that BF-BL circuitry could be very important for generating rhythmic oscillations known to be critical for emotional learning. BF cholinergic inputs to the BNC might also contribute to memory formation by activating M1 receptors located on PN dendritic shafts and spines that also express NMDA receptors.

ß2* nAChR sensitivity modulates acquisition of cocaine self-administration in male rats.

Signaling through nicotinic acetylcholine receptors (nAChRs) plays a role in cocaine reward and reinforcement, suggesting that the cholinergic system could be manipulated with therapeutics to modulate aspects of cocaine use disorder (CUD). We examined the interaction between nAChRs and cocaine reinforcement by expressing a hypersensitive ß2 nAChR subunit (ß2Leu9'Ser) in the ventral tegmental area of male Sprague Dawley rats. Compared to control rats, ß2Leu9'Ser rats acquired (fixed ratio) intravenous cocaine self-administration faster and with greater likelihood. By contrast, ß2Leu9'Ser rats were approximately equivalent to controls in their intake of cocaine on a progressive ratio schedule of reinforcement, suggesting differential effects of cholinergic signaling depending on experimental parameters. Like progressive ratio cocaine SA, ß2Leu9'Ser rats and controls did not differ significantly in food SA assays, including acquisition on a fixed ratio schedule or in progressive ratio sessions. These results highlight the specific role of high-affinity, heteropentameric ß2* (ß2-containing) nAChRs in acquisition of cocaine SA, suggesting that mesolimbic acetylcholine signaling is active during this process.

Evaluation of the risk factors for the failure of a single prophylactic dose of anticholinergic drugs for irinotecan-induced cholinergic symptoms.

OBJECTIVE: Irinotecan (IRI) is an anticancer drug that is frequently used to treat colorectal, gastric, and pancreatic cancers. Its side effects include cholinergic symptoms, such as diarrhea, abdominal pain, nausea, and hyperhidrosis. Anticholinergic medicines are frequently used for treatment or prophylaxis; however, the risk factors for the failure of a single prophylactic anticholinergic administration remain unclear. Moreover, an appropriate anticholinergic drug for prophylaxis remains unknown. Thus, we aimed to identify the risk factors associated with the failure of a single prophylactic dose of anticholinergic drugs for IRI-induced cholinergic symptoms and to evaluate the usefulness of multiple prophylactic doses of anticholinergic drugs. MATERIALS AND METHODS: Patients who underwent IRI treatment for colorectal, gastric, or pancreatic cancer and received prophylactic anticholinergic drugs for IRI-induced cholinergic symptoms (n = 135) were retrospectively evaluated. Univariate and multivariate logistic regression analyses were performed to identify the risk factors for failure of a single prophylactic dose of anticholinergic drugs. We also evaluated the efficacy of multiple prophylactic anticholinergic drug administration. RESULTS: Based on univariate and multivariate analyses, colorectal cancer, female sex, and prophylactic use of scopolamine butyl bromide were identified as risk factors for failure of a single prophylactic dose of anticholinergic drugs. The efficacy of multiple prophylactic doses was confirmed to be 95% of the patients who had a single prophylactic failure due to temporary effect but symptom appearance after a certain period of time (wearing-off). CONCLUSION: We determined that colorectal cancer, female sex, and prophylactic use of scopolamine butyl bromide were risk factors associated with the failure of a single prophylactic dose of anticholinergic drugs, and that multiple prophylactic doses for wearing-off can be a promising method.

Prevention of MK-801-induced amnestic effect with combined activation of 5-HT1A and muscarinic receptors in mice.

BACKGROUND: Muscarinic or 5-HT1A receptors are crucial in learning and memory processes, and their expression is evident in the brain areas involved in cognition. The administration of the activators of these receptors prevents the development of cognitive dysfunctions in animal models of schizophrenia induced by MK-801 (N-methyl-d-aspartate receptor antagonist) administration. GABAergic dysfunction is considered as one of the most important causes of MK-801-induced spatial learning deficits. METHODS: Novel object recognition (NOR) and Morris water maze (MWM) tests were used to study the anti-amnestic effect of the biased 5-HT1A receptor agonist (F15599) alone or in combinations with VU0357017 (M1 receptor allosteric agonist), VU0152100 (M4 receptor positive allosteric modulator), and VU0238429 (M5 receptor positive allosteric modulator) on MK-801-induced dysfunctions. The compounds were administered for 5 consecutive days. Animals tested with the MWM underwent 5-day training. Western blotting was used to study the expressions of 5-HT1A receptors and the level of GAD65 in the frontal cortices (FCs) and hippocampi of the animals. RESULTS: F15599 prevented the amnestic effect induced by MK-801 in the MWM at a dose of 0.1 mg/kg. The co-administration of the compound with muscarinic receptors activators had no synergistic effect. The additive effect of the combinations was evident in the prevention of declarative memory dysfunctions investigated in NOR. The administration of MK-801 impaired 5-HT1A expression in the hippocampi and decreased GAD65 levels in both the FCs and hippocampi. The administration of muscarinic ligands prevented these MK-801-induced deficits only in the hippocampi of MWM-trained animals. No effects of the compounds were observed in untrained mice. CONCLUSION: Our results indicate that F15599 prevents schizophrenia-related spatial learning deficits in the MWM; however, the activity of the compound is not intensified with muscarinic receptors activators. In contrast, the combined administration of the ligands is effective in the NOR model of declarative memory. The muscarinic receptors activators reversed MK-801-induced 5-HT1A and GAD65 dysfunctions in the hippocampi of MWM-trained mice, but not in untrained mice.

Oxidized/unmodified-polyethylene microplastics neurotoxicity in mice: Perspective from microbiota-gut-brain axis.

Microplastics (MPs) are inevitably oxidized in the environment, and their potential toxicity to organisms has attracted wide attention. However, the neurotoxicity and mechanism of oxidized polyethylene (Ox-PE) MPs to organisms remain unclear. Herein, we prepared oxidized low-density polyethylene (Ox-LDPE) and established a model of MPs exposure by continuously orally gavage of C57BL/6 J mice with LDPE-MPs/Ox-LDPE-MPs for 28 days with or without oral administration of Lactobacillus plantarum DP189 and galactooligosaccharides (DP189&GOS). The experimental results indicated that LDPE-MPs or Ox-LDPE-MPs caused several adverse effects in mice, mainly manifested by behavioral changes, disruption of the intestinal and blood-brain barrier (BBB), and simultaneous oxidative stress, inflammatory reactions, and pathological damage in the brain and intestines. Brain transcriptomic analysis revealed that the cholinergic synaptic signaling pathways, which affect cognitive function, were significantly disrupted after exposure to LDPE-MPs or Ox-LDPE-MPs. Real-time quantitative polymerase chain reaction and Western Blotting results further demonstrated that the critical genes (Slc5a7, Chat and Slc18a3) and proteins (Chat and Slc18a3) in the cholinergic synaptic signaling pathway were significantly down-regulated after exposure to LDPE-MPs or Ox-LDPE-MPs. These alterations lead to reduced acetylcholine concentration, which causes cognitive dysfunction in mice. Importantly, the DP189&GOS interventions effectively mitigated the MPs-induced cognitive dysfunction and intestinal microbiota alteration, improved intestinal and BBB integrity, attenuated the oxidative stress and inflammatory response, and also saw a rebound in the release of acetylcholine. These results indicated that LDPE-MPs and Ox-LDPE-MPs exert neurotoxic effects on mice by inducing oxidative stress, inflammatory responses, and dysregulation of cholinergic signaling pathways in the mouse brain. That probiotic supplementation is effective in attenuating MPs-induced neurotoxicity in mice. Overall, this study reveals the potential mechanisms of neurotoxicity of LDPE-MPs and Ox-LDPE-MPs on mice and their improvement measures, necessary to assess the potential risks of plastic contaminants to human health.

Exogenous monosodium glutamate exacerbates lipopolysaccharide-induced neurobehavioral deficits, oxidative damage, neuroinflammation, and cholinergic dysfunction in rat brain.

Extensive experimental evidence points to neuroinflammation and oxidative stress as major pathogenic events that initiate and drive the neurodegenerative process. Monosodium glutamate (MSG) is a widely used food additive in processed foods known for its umami taste-enhancing properties. However, concerns about its potential adverse effects on the brain have been raised. Thus, the present study investigated the impact of MSG on lipopolysaccharide (LPS)-induced neurotoxicity in rat brains. Wistar rats weighing between 180 g and 200 g were randomly allocated into four groups: control (received distilled water), MSG (received 1.5 g/kg/day), LPS (received 250 µg/kg/day), and LPS + MSG (received LPS, 250 µg/kg, and MSG, 1.5 g/kg). LPS was administered intraperitoneally for 7 days while MSG was administered orally for 14 days. Our results showed that MSG exacerbated LPS-induced impairment in locomotor and exploratory activities in rats. Similarly, MSG exacerbated LPS-induced oxidative stress as evidenced by increased levels of malondialdehyde (MDA) with a concomitant decrease in levels of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione-s-transferase (GST) in the brain tissue. In addition, MSG potentiated LPS-induced neuroinflammation, as indicated by increased levels of pro-inflammatory cytokines such as interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) as well as myeloperoxidase (MPO) and nitric oxide (NO) in the brain. Moreover, MSG aggravated LPS-induced cholinergic dysfunction, as demonstrated by increased activity of acetylcholinesterase (AChE) in the brain. Further, we found a large number of degenerative neurons widespread in hippocampal CA1, CA3 regions, cerebellum, and cortex according to H&E staining. Taken together, our findings suggest that MSG aggravates LPS-induced neurobehavioral deficits, oxidative stress, neuroinflammation, cholinergic dysfunction, and neurodegeneration in rat brains.

Aspiration removal of orbitofrontal cortex disrupts cholinergic fibers of passage to anterior cingulate cortex in rhesus macaques.

The study of anthropoid nonhuman primates has provided valuable insights into frontal cortex function in humans, as these primates share similar frontal anatomical subdivisions (Murray et al. 2011). Causal manipulation studies have been instrumental in advancing our understanding of this area. One puzzling finding is that macaques with bilateral aspiration removals of orbitofrontal cortex (OFC) are impaired on tests of cognitive flexibility and emotion regulation, whereas those with bilateral excitotoxic lesions of OFC are not (Rudebeck et al. 2013). This discrepancy is attributed to the inadvertent disruption of fibers of passage by aspiration lesions but not by excitotoxic lesions. Which fibers of passage are responsible for the impairments observed? One candidate is cholinergic fibers originating in the nucleus basalis magnocellularis (NBM) and passing nearby or through OFC on their way to other frontal cortex regions (Kitt et al. 1987). To investigate this possibility, we performed unilateral aspiration lesions of OFC in three macaques, and then compared cholinergic innervation of the anterior cingulate cortex (ACC) between hemispheres. Histological assessment revealed diminished cholinergic innervation in the ACC of hemispheres with OFC lesions relative to intact hemispheres. This finding indicates that aspiration lesions of the OFC disrupt cholinergic fibers of passage, and suggests the possibility that loss of cholinergic inputs to ACC contributes to the impairments in cognitive flexibility and emotion regulation observed after aspiration but not excitotoxic lesions of OFC.

EDITOR SPOTLIGHT: Interview with Cholinergic Mechanisms Special Issue Guest Editor Hermona Soreq.

Hermona Soreq holds a Hebrew University Slesinger Chair in Molecular Neuroscience and is among the founding members of the Edmond and Lily Safra Center of Brain Sciences (ELSC). Soreq's research (H-impact: 98) focuses on acetylcholine (ACh)-related pathways and combines RNA-sequencing technologies, transgenic engineering, and molecular biology tests with in-depth analysis approaches. Her work addresses microRNAs (miRs) and transfer RNA fragments (tRFs) which have rapidly acquired wide recognition as global controllers of regulatory processes in healthy and diseased brain and body, including anxiety, inflammation, and cognition. Altogether, Soreq's work leads to molecular neuroscience-driven prevention and/or intervention with diseases involving impaired ACh signaling, including schizophrenia, bipolar disorder, Alzheimer's disease, and stress. Hermona led this Special Issue based on the 17th Symposium on Cholinergic Mechanisms (ISCM2022). We interviewed her on the progress in the field, what she wants to achieve as Senior Editor for the Gene Regulation and Genetics category at the Journal of Neurochemistry, key moments, and future directions.

Influence of age on nicotinic cholinergic regulation of blood flow in rat's olfactory bulb and neocortex.

The olfactory bulb receives cholinergic basal forebrain inputs as does the neocortex. With a focus on nicotinic acetylcholine receptors (nAChRs), this review article provides an overview and discussion of the following findings: (1) the nAChRs-mediated regulation of regional blood flow in the neocortex and olfactory bulb, (2) the nAChR subtypes that mediate their responses, and (3) their activity in old rats. The activation of the α4ß2-like subtype of nAChRs produces vasodilation in the neocortex, and potentiates olfactory bulb vasodilation induced by olfactory stimulation. The nAChR activity producing neocortical vasodilation was similarly maintained in 2-year-old rats as in adult rats, but was clearly reduced in 3-year-old rats. In contrast, nAChR activity in the olfactory bulb was reduced already in 2-year-old rats. Thus, age-related impairment of α4ß2-like nAChR function may occur earlier in the olfactory bulb than in the neocortex. Given the findings, the vasodilation induced by α4ß2-like nAChR activation may be beneficial for neuroprotection in the neocortex and the olfactory bulb.

Sleep deprivation drives brain-wide changes in cholinergic presynapse abundance in Drosophila melanogaster.

Sleep is an evolutionarily conserved state that supports brain functions, including synaptic plasticity, in species across the animal kingdom. Here, we examine the neuroanatomical and cell-type distribution of presynaptic scaling in the fly brain after sleep loss. We previously found that sleep loss drives accumulation of the active zone scaffolding protein Bruchpilot (BRP) within cholinergic Kenyon cells of the Drosophila melanogaster mushroom body (MB), but not in other classes of MB neurons. To test whether similar cell type-specific trends in plasticity occur broadly across the brain, we used a flp-based genetic reporter to label presynaptic BRP in cholinergic, dopaminergic, GABAergic, or glutamatergic neurons. We then collected whole-brain confocal image stacks of BRP intensity to systematically quantify BRP, a marker of presynapse abundance, across 37 neuropil regions of the central fly brain. Our results indicate that sleep loss, either by overnight (12-h) mechanical stimulation or chronic sleep disruption in insomniac mutants, broadly elevates cholinergic synapse abundance across the brain, while synapse abundance in neurons that produce other neurotransmitters undergoes weaker, if any, changes. Extending sleep deprivation to 24 h drives brain-wide upscaling in glutamatergic, but not other, synapses. Finally, overnight male-male social pairings induce increased BRP in excitatory synapses despite male-female pairings eliciting more waking activity, suggesting experience-specific plasticity. Within neurotransmitter class and waking context, BRP changes are similar across the 37 neuropil domains, indicating that similar synaptic scaling rules may apply across the brain during acute sleep loss and that sleep need may broadly alter excitatory-inhibitory balance in the central brain.

Role of M4 -receptor cholinergic signaling in direct pathway striatal projection neurons during dopamine depletion.

Direct pathway striatal projection neurons (dSPNs) are characterized by the expression of dopamine (DA) class 1 receptors (D1 R), as well as cholinergic muscarinic M1 and M4 receptors (M1 R, M4 R). D1 R enhances neuronal firing through phosphorylation of voltage-gate calcium channels (CaV 1 Ca2+ channels) activating Gs proteins and protein kinase A (PKA). Concurrently, PKA suppresses phosphatase PP-1 through DARPP-32, thus extending this facilitatory modulation. M1 R also influences Ca2+ channels in SPNs through Gq proteins and protein kinase C. However, the signaling mechanisms of M4 R in dSPNs are less understood. Two pathways are attributed to M4 R: an inhibitory one through Gi/o proteins, and a facilitatory one via the cyclin Cdk5. Our study reveals that a previously observed facilitatory modulation via CaV 1 Ca2+ channels is linked to the Cdk5 pathway in dSPNs. This result could be significant in treating parkinsonism. Therefore, we questioned whether this effect persists post DA-depletion in experimental parkinsonism. Our findings indicate that in such conditions, M4 R activation leads to a decrease in Ca2+ current and an increased M4 R protein level, contrasting with the control response. Nevertheless, parkinsonian and control actions are inhibited by the Cdk5 inhibitor roscovitine, suggesting Cdk5's role in both conditions. Cdk5 may activate PP-1 via PKA inhibition in DA depletion. Indeed, we found that inhibiting PP-1 restores control M4 R actions, implying that PP-1 is overly active via M4 Rs in DA-depleted condition. These insights contribute to understanding how DA-depletion alters modulatory signaling in striatal neurons. Additional working hypotheses are discussed.

Cholinergic Receptor Muscarinic 1 Co-Localized with Mitochondria in Cultured Dorsal Root Ganglion Neurons, and Its Deletion Disrupted Mitochondrial Ultrastructure in Peripheral Neurons: Implications in Alzheimer's Disease.

Background: Loss of Cholinergic Receptor Muscarinic 1 (CHRM1) has been linked to the pathogenesis of Alzheimer's disease (AD). Our recent study found significantly lower CHRM1 protein levels in AD patient cortices, linked to reduced survival. Furthermore, using knockout mice (Chrm1-/-) we demonstrated that deletion of Chrm1 alters cortical mitochondrial structure and function, directly establishing a connection between its loss and mitochondrial dysfunction in the context of AD. While CHRM1's role in the brain has been extensively investigated, its impact on peripheral neurons in AD remains a crucial area of research, especially considering reported declines in peripheral nerve conduction among AD patients. Objective: The objective was to characterize Chrm1 localization and mitochondrial deficits in Chrm1-/- dorsal root ganglion (DRG) neurons. Methods: Recombinant proteins tagged with Green or Red Fluorescent Protein (GFP/RFP) were transiently expressed to investigate the localization of Chrm1 and mitochondria, as well as mitochondrial movement in the neurites of cultured primary mouse DRG neurons, using confocal time-lapse live cell imaging. Transmission electron microscopy was performed to examine the ultrastructure of mitochondria in both wild-type and Chrm1-/- DRGs. Results: Fluorescence imaging revealed colocalization and comigration of N-terminal GFP-tagged Chrm1 and mitochondrial localization signal peptide-tagged RFP-labelled mitochondria in the DRGs neurons. A spectrum of mitochondrial structural abnormalities, including disruption and loss of cristae was observed in 87% neurons in Chrm1-/- DRGs. Conclusions: This study suggests that Chrm1 may be localized in the neuronal mitochondria and loss of Chrm1 in peripheral neurons causes sever mitochondrial structural aberrations resembling AD pathology.

Cholinesterase activities and sepsis-associated encephalopathy in viral versus nonviral sepsis.

PURPOSE: There is evidence that cholinergic imbalance secondary to neuroinflammation plays a role in the pathophysiology of sepsis-associated encephalopathy (SAE). Blood acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities have been proposed as surrogate parameters for the cholinergic function of the central nervous system. Viral sepsis is associated with systemic inflammation and BChE has been reported to be of prognostic value in a small cohort of COVID-19 patients. Nevertheless, the prognostic value of AChE in patients with viral sepsis remains unclear. METHODS: We investigated the role of AChE and BChE activities as prognostic biomarkers of SAE and mortality in patients with viral vs nonviral sepsis enrolled in two prospective cohort studies. We quantified the AChE and BChE activities in whole blood of patients at two time points in the acute phase of viral sepsis (N = 108) and compared them with the activities in patients with nonviral sepsis (N = 117) and healthy volunteers (N = 81). Patients were observed until discharge from the intensive care unit (ICU). RESULTS: Three days after sepsis onset, the median [interquartile range] levels of AChE and BChE were reduced in both patients with viral sepsis (AChE, 5,105 [4,010-6,250] U·L-1; BChE, 1,943 [1,393-2,468] U·L-1) and nonviral sepsis (AChE, 4,424 [3,630-5,055] U·L-1; BChE, 1,095 [834-1,526] U·L-1) compared with healthy volunteers (AChE, 6,693 [5,401-8,020] U·L-1; BChE, 2,645 [2,198-3,478] U·L-1). Patients with viral sepsis with SAE during their ICU stay had lower AChE activity three days after sepsis onset than patients without SAE (4,249 [3,798-5,351] U·L-1 vs 5,544 [4,124-6,461] U·L-1). Butyrylcholinesterase activity seven days after sepsis onset was lower in patients with viral sepsis who died in the ICU than in surviving patients (1,427 [865-2,181] U·L-1 vs 2,122 [1,571-2,787] U·L-1). CONCLUSION: Cholinesterase activities may be relevant prognostic markers for the occurrence of SAE and mortality in the ICU in patients with viral sepsis. STUDY REGISTRATION: This study constitutes an analysis of data from the ongoing studies ICROS (NCT03620409, first submitted 15 May 2018) and ICROVID (DRKS00024162, first submitted 9 February 2021).

RéSUMé: OBJECTIF: Certaines données probantes soutiennent que le déséquilibre cholinergique secondaire à la neuroinflammation joue un rôle dans la physiopathologie de l'encéphalopathie associée au sepsis (EAS). Les activités de l'acétylcholinestérase (AChE) et de la butyrylcholinestérase (BChE) sanguines ont été proposées comme paramètres de substitution de la fonction cholinergique du système nerveux central. Le sepsis viral est associé à une inflammation systémique et il a été rapporté que la BChE possédait une valeur pronostique dans une petite cohorte atteinte de COVID-19. Néanmoins, la valeur pronostique de l'AChE chez les patient·es atteint·es de sepsis viral reste incertaine. MéTHODE: Nous avons étudié le rôle des activités de l'AChE et de la BChE en tant que biomarqueurs pronostiques de l'EAS et de la mortalité chez les patient·es atteint·es de sepsis viral vs non viral recruté·es dans deux études de cohorte prospectives. Nous avons quantifié les activités de l'AChE et de la BChE dans le sang total de patient·es à deux moments de la phase aiguë du sepsis viral (N = 108) et les avons comparées aux activités chez les patient·es atteint·es de sepsis non viral (N = 117) et chez des volontaires sain·es (N = 81). Les patient·es ont été observé·es jusqu'à leur sortie de l'unité de soins intensifs (USI). RéSULTATS: Trois jours après l'apparition du sepsis, les taux médians [écart interquartile] d'AChE et BChE étaient réduits tant chez la patientèle atteinte de sepsis viral (AChE, 5105 [4010­6250] U·L−1; BChE, 1943 [1393­2468] U·L−1) et de sepsis non viral (AChE, 4424 [3630­5055] U·L−1; BChE, 1095 [834­1526] U·L−1) par rapport aux volontaires sain·es (AChE, 6693 [5401­8020] U·L−1; BChE, 2645 [2198­3478] U·L−1). Les patient·es atteint·es de sepsis viral avec EAS pendant leur séjour aux soins intensifs avaient une activité AChE plus faible trois jours après l'apparition du sepsis que les personnes sans EAS (4249 [3798­5351] U·L−1 vs 5544 [4124­6461] U·L−1). L'activité de la butyrylcholinestérase sept jours après l'apparition du sepsis était plus faible chez les patient·es atteint·es de sepsis viral décédé·es à l'USI que chez les personnes ayant survécu (1427 [865­2181] U·L-1 vs 2122 [1571­2787] U·L-1). CONCLUSION: Les activités des cholinestérases pourraient constituer des marqueurs pronostiques pertinents pour la survenue d'EAS et la mortalité en soins intensifs chez la patientèle atteinte de sepsis viral. ENREGISTREMENT DE L'éTUDE: Cette étude constitue une analyse des données des études en cours ICROS (NCT03620409, première soumission le 15 mai 2018) et ICROVID (DRKS00024162, première soumission le 9 février 2021).

Taming Microglia in Alzheimer's Disease: Exploring Potential Implications of Choline Alphoscerate via α7 nAChR Modulation.

Alzheimer's disease (AD), marked by cognitive impairment, predominantly affects the brain regions regulated by cholinergic innervation, such as the cerebral cortex and hippocampus. Cholinergic dysfunction, a key contributor to age-related cognitive decline, has spurred investigations into potential therapeutic interventions. We have previously shown that choline alphoscerate (α-GPC), a cholinergic neurotransmission-enhancing agent, protects from Aß-mediated neurotoxicity. Herein, we investigated the effects of α-GPC on the microglial phenotype in response to Aß via modulation of the nicotinic alpha-7 acetylcholine receptor (α7 nAChR). BV2 microglial cells were pre-treated for 1 h with α-GPC and were treated for 24, 48, and 72 h with Aß1-42 and/or α-BTX, a selective α7nAchR antagonist. Fluorescent immunocytochemistry and Western blot analysis showed that α-GPC was able to antagonize Aß-induced inflammatory effects. Of note, α-GPC exerted its anti-inflammatory effect by directly activating the α7nAChR receptor, as suggested by the induction of an increase in [Ca2+]i and Ach-like currents. Considering that cholinergic transmission appears crucial in regulating the inflammatory profiles of glial cells, its modulation emerges as a potential pharmaco-therapeutic target to improve outcomes in inflammatory neurodegenerative disorders, such as AD.

Cell-type-specific optogenetic fMRI on basal forebrain reveals functional network basis of behavioral preference.

The basal forebrain (BF) is a complex structure that plays key roles in regulating various brain functions. However, it remains unclear how cholinergic and non-cholinergic BF neurons modulate large-scale functional networks and their relevance in intrinsic and extrinsic behaviors. With an optimized awake mouse optogenetic fMRI approach, we revealed that optogenetic stimulation of four BF neuron types evoked distinct cell-type-specific whole-brain BOLD activations, which could be attributed to BF-originated low-dimensional structural networks. Additionally, optogenetic activation of VGLUT2, ChAT, and PV neurons in the BF modulated the preference for locomotion, exploration, and grooming, respectively. Furthermore, we uncovered the functional network basis of the above BF-modulated behavioral preference through a decoding model linking the BF-modulated BOLD activation, low-dimensional structural networks, and behavioral preference. To summarize, we decoded the functional network basis of differential behavioral preferences with cell-type-specific optogenetic fMRI on the BF and provided an avenue for investigating mouse behaviors from a whole-brain view.