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.

Striato-cortical functional connectivity changes in mild cognitive impairment with Lewy bodies.

BACKGROUND: Functional connectivity changes in clinically overt neurodegenerative diseases such as dementia with Lewy bodies have been described, but studies on connectivity changes in the pre-dementia phase are scarce. OBJECTIVES: We concentrated on evaluating striato-cortical functional connectivity differences between patients with Mild Cognitive Impairment with Lewy bodies and healthy controls and on assessing the relation to cognition. METHODS: Altogether, we enrolled 77 participants (47 patients, of which 35 met all the inclusion criteria for the final analysis, and 30 age- and gender-matched healthy controls, of which 28 met all the inclusion criteria for the final analysis) to study the seed-based connectivity of the dorsal, middle, and ventral striatum. We assessed correlations between functional connectivity in the regions of between-group differences and neuropsychological scores of interest (visuospatial and executive domains z-scores). RESULTS: Subjects with Mild Cognitive Impairment with Lewy Bodies, as compared to healthy controls, showed increased connectivity from the dorsal part of the striatum particularly to the bilateral anterior part of the temporal cortex with an association with executive functions. CONCLUSIONS: We were able to capture early abnormal connectivity within cholinergic and noradrenergic pathways that correlated with cognitive functions known to be linked to cholinergic/noradrenergic deficits. The knowledge of specific alterations may improve our understanding of early neural changes in pre-dementia stages and enhance research of disease modifying therapy.

Defining morphologically and genetically distinct GABAergic/cholinergic amacrine cell subtypes in the vertebrate retina.

In mammals, retinal direction selectivity originates from GABAergic/cholinergic amacrine cells (ACs) specifically expressing the sox2 gene. However, the cellular diversity of GABAergic/cholinergic ACs of other vertebrate species remains largely unexplored. Here, we identified 2 morphologically and genetically distinct GABAergic/cholinergic AC types in zebrafish, a previously undescribed bhlhe22+ type and a mammalian counterpart sox2+ type. Notably, while sole sox2 disruption removed sox2+ type, the codisruption of bhlhe22 and bhlhe23 was required to remove bhlhe22+ type. Also, both types significantly differed in dendritic arbors, lamination, and soma position. Furthermore, in vivo two-photon calcium imaging and the behavior assay suggested the direction selectivity of both AC types. Nevertheless, the 2 types showed preferential responses to moving bars of different sizes. Thus, our findings provide new cellular diversity and functional characteristics of GABAergic/cholinergic ACs in the vertebrate retina.

Vitamin B12 produced by gut bacteria modulates cholinergic signalling.

A growing body of evidence indicates that gut microbiota influence brain function and behaviour. However, the molecular basis of how gut bacteria modulate host nervous system function is largely unknown. Here we show that vitamin B12-producing bacteria that colonize the intestine can modulate excitatory cholinergic signalling and behaviour in the host Caenorhabditis elegans. Here we demonstrate that vitamin B12 reduces cholinergic signalling in the nervous system through rewiring of the methionine (Met)/S-adenosylmethionine cycle in the intestine. We identify a conserved metabolic crosstalk between the methionine/S-adenosylmethionine cycle and the choline-oxidation pathway. In addition, we show that metabolic rewiring of these pathways by vitamin B12 reduces cholinergic signalling by limiting the availability of free choline required by neurons to synthesize acetylcholine. Our study reveals a gut-brain communication pathway by which enteric bacteria modulate host behaviour and may affect neurological health.

Aflatoxin B1-induced redox imbalance in the hippocampus and cerebral cortex of male Wistar rats is accompanied by altered cholinergic, indoleaminergic, and purinergic pathways: Abatement by dietary rutin.

The neurotoxic impact of dietary exposure to aflatoxin B1 (AFB1) is well documented in experimental studies. Rutin is a phytochemical with prominent anti-inflammatory and antioxidant activities. There is an information gap on the influence of rutin on AFB1-induced neurotoxicity. This study investigated the influence of rutin on neurobehavioral and biochemical abnormalities in male Wistar rats (six weeks old) orally treated with AFB1 (0.75, and 1.5 mg/kg body weight) or co-administered with rutin (50 mg/kg) for 30 uninterrupted days. Results indicate that AFB1-induced depression-like behavior by Tail Suspension Test (TST) and cognitive impairment by Y-maze was abated following rutin co-administration. Abatement of AFB1-induced decreases in acetylcholinesterase (AChE) activity, and increased antioxidant status, by rutin was accompanied by a marked reduction in oxidative stress markers and increased hydrolysis of the purinergic molecules in the cerebral cortex and hippocampus of rats. Additionally, rutin co-treatment abrogated AFB1-mediated elevation of interleukin-6 (IL-6), nitric oxide (NO) levels, and activity of myeloperoxidase (MPO). Correspondingly, rutin co-treatment lowered the activity and immunocontent of immunosuppressive indoleamine 2, 3-dioxygenase (IDO). Further, rutin co-treatment prevented histological injuries in the cerebral cortex and hippocampus. In conclusion, abatement of AFB1-induced neurobehavioral abnormalities by rutin involves the mechanisms of anti-inflammatory, antioxidant, and regulation of cholinergic, purinergic, and indoleaminergic pathways in rats.

Cholinergic signaling via the α7 nicotinic acetylcholine receptor regulates the migration of monocyte-derived macrophages during acute inflammation.

BACKGROUND: The involvement of the autonomic nervous system in the regulation of inflammation is an emerging concept with significant potential for clinical applications. Recent studies demonstrate that stimulating the vagus nerve activates the cholinergic anti-inflammatory pathway that inhibits pro-inflammatory cytokines and controls inflammation. The α7 nicotinic acetylcholine receptor (α7nAChR) on macrophages plays a key role in mediating cholinergic anti-inflammatory effects through a downstream intracellular mechanism involving inhibition of NF-κB signaling, which results in suppression of pro-inflammatory cytokine production. However, the role of the α7nAChR in the regulation of other aspects of the immune response, including the recruitment of monocytes/macrophages to the site of inflammation remained poorly understood. RESULTS: We observed an increased mortality in α7nAChR-deficient mice (compared with wild-type controls) in mice with endotoxemia, which was paralleled with a significant reduction in the number of monocyte-derived macrophages in the lungs. Corroborating these results, fluorescently labeled α7nAChR-deficient monocytes adoptively transferred to WT mice showed significantly diminished recruitment to the inflamed tissue. α7nAChR deficiency did not affect monocyte 2D transmigration across an endothelial monolayer, but it significantly decreased the migration of macrophages in a 3D fibrin matrix. In vitro analysis of major adhesive receptors (L-selectin, ß1 and ß2 integrins) and chemokine receptors (CCR2 and CCR5) revealed reduced expression of integrin αM and αX on α7nAChR-deficient macrophages. Decreased expression of αMß2 was confirmed on fluorescently labeled, adoptively transferred α7nAChR-deficient macrophages in the lungs of endotoxemic mice, indicating a potential mechanism for α7nAChR-mediated migration. CONCLUSIONS: We demonstrate a novel role for the α7nAChR in mediating macrophage recruitment to inflamed tissue, which indicates an important new aspect of the cholinergic regulation of immune responses and inflammation.

Subregion and sex differences in ethanol activation of cholinergic and glutamatergic cells in the mesopontine tegmentum.

Ethanol engages cholinergic signaling and elicits endogenous acetylcholine release. Acetylcholine input to the midbrain originates from the mesopontine tegmentum (MPT), which is composed of the laterodorsal tegmentum (LDT) and the pedunculopontine tegmental nucleus (PPN). We investigated the effect of acute and chronic ethanol administration on cholinergic and glutamatergic neuron activation in the PPN and LDT in male and female mice. We show that ethanol activates neurons of the PPN and not the LDT in male mice. Chronic 15 daily injections of 2 g/kg ethanol induced Fos expression in cholinergic and glutamatergic PPN neurons in male mice, whereas ethanol did not increase cholinergic and glutamatergic neuronal activation in the LDT. A single acute 4 g/kg injection, but not a single 2 g/kg injection, induced cholinergic neuron activation in the male PPN but not the LDT. In contrast, acute or chronic ethanol at either dose or duration had no effect on the activation of cholinergic or glutamatergic neurons in the MPT of female mice. Female mice had higher baseline level of activation in cholinergic neurons compared with males. We also found a population of co-labeled cholinergic and glutamatergic neurons in the PPN and LDT which were highly active in the saline- and ethanol-treated groups in both sexes. These findings illustrate the complex differential effects of ethanol across dose, time point, MPT subregion and sex.

Cellular effects of microplastics are influenced by their dimension: Mechanistic relationships and integrated criteria for particles definition.

The definition of microplastics (MPs) is nowadays too generic from a biological perspective, since different characteristics of these particles might influence their effects. To provide experimental evidence that size is an important factor to be considered, Mediterranean mussels Mytilus galloprovincialis were exposed to five size classes of polyethylene fragments (PE-MPs, 20-50 µm, 50-100 µm, 100-250 µm, 250-500 µm, 500-1000 µm). After 10 days of exposure, MPs ingestion and mechanistic relationships between particles size and cellular effects were analysed through a wide panel of biological alterations, including immune system responses, cholinergic function, antioxidant system, lipid metabolism and peroxidation. Results were further elaborated through a Weight of Evidence approach, summarizing the overall biological significance of obtained results in a hazard index based on the number and magnitude of variations and their toxicological relevance. PE-MPs 500-1000 µm were identified as the less biologically reactive size class due to the limited ingestion of particles coupled with the lack of biological effects, followed by PE-MPs 250-500 µm, which slightly altered the cholinergic function and lysosomal membranes. Conversely, PE-MPs smaller than 250 µm provoked a more consistent onset of biological alterations in terms of immune system composition and functioning, redox homeostasis, and lipid metabolism. The overall findings of this study highlight the importance of considering the size of particles for monitoring and risk assessment of MPs, introducing a more integrated evaluation of plastic pollution that, beside particles concentration, should adequately weigh those characteristics triggering the onset of biological effects.

Stimulation of the muscarinic receptor M4 regulates neural precursor cell proliferation and promotes adult hippocampal neurogenesis.

Cholinergic signaling plays a crucial role in the regulation of adult hippocampal neurogenesis; however, the mechanisms by which acetylcholine mediates neurogenic effects are not completely understood. Here, we report the expression of muscarinic acetylcholine receptor subtype M4 (M4 mAChR) on a subpopulation of neural precursor cells (NPCs) in the adult mouse hippocampus, and demonstrate that its pharmacological stimulation promotes their proliferation, thereby enhancing the production of new neurons in vivo. Using a targeted ablation approach, we also show that medial septum (MS) and the diagonal band of Broca (DBB) cholinergic neurons support both the survival and morphological maturation of adult-born neurons in the mouse hippocampus. Although the systemic administration of an M4-selective allosteric potentiator fails to fully rescue the MS/DBB cholinergic lesion-induced decrease in hippocampal neurogenesis, it further exacerbates the impairment in the morphological maturation of adult-born neurons. Collectively, these findings reveal stage-specific roles of M4 mAChRs in regulating adult hippocampal neurogenesis, uncoupling their positive role in enhancing the production of new neurons from the M4-induced inhibition of their morphological maturation, at least in the context of cholinergic signaling dysfunction.

Assessing the effect of breed, age, and sex on muscarinic receptor distribution in atria and ventricles of turkeys.

Atrial and ventricular myocardium from young (6-wk-old), young adult (3-6-mo-old), and aged (14-15-mo-old) meat-type (B.U.T. Big 6) and wild-type (Cröllwitzer) turkeys were used to study the influence of age and sex on cholinergic muscarinic receptors using [3H]-N-methyl-scopolamine (3H-NMS) binding studies. In both breeds, saturation experiments indicated the presence of regional-, sex-, and age-related differences in the density of cholinergic muscarinic receptors (Bmax), that is, a decrease or increase. Except for right atria, Bmax was decreased in both male and female B.U.T. Big 6 hearts with increasing age. Similarly, a negative correlation between Bmax and age could be seen in female and male atria of Cröllwitzer turkeys, while positive correlation could be seen in right and left ventricles of male, and only right ventricles of female Cröllwitzer turkeys. The affinity of the receptor (KD) was not affected by age, sex and breed. In all cardiac chamber tissues, the M2-subtype was shown to be predominant followed by the M3-subtype and to a lesser extent the M1-subtype. Aspects of this age-dependent remodeling of the heart differ between sexes, resulting in maladaptive changes in older turkeys with a high degree of frailty. These observations may help explain why males and females are susceptible to different cardiovascular diseases as they age and why frail older adults are most often affected by these diseases.

Environmental levels of azoxystrobin disturb male zebrafish behavior: Possible roles of oxidative stress, cholinergic system, and dopaminergic system.

A widely applied pesticide of azoxystrobin, is increasingly detected in the water environment. Concern has been raised against its potential detriment to aquatic ecosystems. It has been shown that exposure to azoxystrobin interfere with the locomotor behavior of zebrafish larvae. This study aims to investigate whether exposure to environmental levels of azoxystrobin (2 µg/L, 20 µg/L, and 200 µg/L) changes the behavior of male adult zebrafish. Herein, we evaluated behavioral response (locomotor, anxiety-like, and exploratory behaviors), histopathology, biochemical indicators, and gene expression in male adult zebrafish upon azoxystrobin exposure. The study showed that exposure to azoxystrobin for 42 days remarkably increased the locomotor ability of male zebrafish, resulted in anxiety-like behavior, and inhibited exploratory behavior. After treatment with 200 µg/L azoxystrobin, vasodilatation, and congestion were observed in male zebrafish brains. Exposure to 200 µg/L azoxystrobin notably elevated ROS level, MDA concentration, CAT activity, and AChE activity, while inhibiting SOD activity, GPx activity, ACh concentration, and DA concentration in male zebrafish brains. Moreover, the expression levels of genes related to the antioxidant, cholinergic, and dopaminergic systems were significantly changed. This suggests that azoxystrobin may interfere with the homeostasis of neurotransmitters by causing oxidative stress in male zebrafish brains, thus affecting the behavioral response of male zebrafish.

Activation of a non-neuronal cholinergic system in visceral white adipose tissue of obese mice and humans.

BACKGROUND AND OBJECTIVES: Since white adipose tissue (WAT) lacks parasympathetic cholinergic innervation, the source of the acetylcholine (ACh) acting on white adipocyte cholinergic receptors is unknown. This study was designed to identify ACh-producing cells in mouse and human visceral WAT and to determine whether a non-neuronal cholinergic system becomes activated in obese inflamed WAT. METHODS: Mouse epididymal WAT (eWAT) and human omental fat were studied in normal and obese subjects. The expression of the key molecules involved in cholinergic signaling was evaluated by qRT-PCR and western blotting whereas their tissue distribution and cellular localization were investigated by immunohistochemistry, confocal microscopy and in situ hybridization. ACh levels were measured by liquid chromatography/tandem mass spectrometry. The cellular effects of ACh were assessed in cultured human multipotent adipose-derived stem cell (hMADS) adipocytes. RESULTS: In mouse eWAT, diet-induced obesity modulated the expression of key cholinergic molecular components and, especially, raised the expression of choline acetyltransferase (ChAT), the ACh-synthesizing enzyme, which was chiefly detected in interstitial macrophages, in macrophages forming crown-like structures (CLSs), and in multinucleated giant cells (MGCs). The stromal vascular fraction of obese mouse eWAT contained significantly higher ACh and choline levels than that of control mice. ChAT was undetectable in omental fat from healthy subjects, whereas it was expressed in a number of interstitial macrophages, CLSs, and MGCs from some obese individuals. In hMADS adipocytes stressed with tumor necrosis factor α, ACh, alone or combined with rivastigmine, significantly blunted monocyte chemoattractant protein 1 and interleukin 6 expression, it partially but significantly, restored adiponectin and GLUT4 expression, and promoted glucose uptake. CONCLUSIONS: In mouse and human visceral WAT, obesity induces activation of a macrophage-dependent non-neuronal cholinergic system that is capable of exerting anti-inflammatory and insulin-sensitizing effects on white adipocytes.

Cholinergic Polarization of Human Macrophages.

Macrophages serve as vital defenders, protecting the body by exhibiting remarkable cellular adaptability in response to invading pathogens and various stimuli. These cells express nicotinic acetylcholine receptors, with the α7-nAChR being extensively studied due to its involvement in activating the cholinergic anti-inflammatory pathway. Activation of this pathway plays a crucial role in suppressing macrophages' production of proinflammatory cytokines, thus mitigating excessive inflammation and maintaining host homeostasis. Macrophage polarization, which occurs in response to specific pathogens or insults, is a process that has received limited attention concerning the activation of the cholinergic anti-inflammatory pathway and the contributions of the α7-nAChR in this context. This review aims to present evidence highlighting how the cholinergic constituents in macrophages, led by the α7-nAChR, facilitate the polarization of macrophages towards anti-inflammatory phenotypes. Additionally, we explore the influence of viral infections on macrophage inflammatory phenotypes, taking into account cholinergic mechanisms. We also review the current understanding of macrophage polarization in response to these infections. Finally, we provide insights into the relatively unexplored partial duplication of the α7-nAChR, known as dup α7, which is emerging as a significant factor in macrophage polarization and inflammation scenarios.

Pathway-specific alterations in striatal excitability and cholinergic modulation in a SAPAP3 mouse model of compulsive motor behavior.

Deletion of the obsessive-compulsive disorder (OCD)-associated gene SAP90/PSD-95-associated protein 3 (Sapap3), which encodes a postsynaptic anchoring protein at corticostriatal synapses, causes OCD-like motor behaviors in mice. While corticostriatal synaptic dysfunction is central to this phenotype, the striatum efficiently adapts to pathological changes, often in ways that expand upon the original circuit impairment. Here, we show that SAPAP3 deletion causes non-synaptic and pathway-specific alterations in dorsolateral striatum circuit function. While somatic excitability was elevated in striatal projection neurons (SPNs), dendritic excitability was exclusively enhanced in direct pathway SPNs. Layered on top of this, cholinergic modulation was altered in opposing ways: striatal cholinergic interneuron density and evoked acetylcholine release were elevated, while basal muscarinic modulation of SPNs was reduced. These data describe how SAPAP3 deletion alters the striatal landscape upon which impaired corticostriatal inputs will act, offering a basis for how pathological synaptic integration and unbalanced striatal output underlying OCD-like behaviors may be shaped.

Human airway tuft cells influence the mucociliary clearance through cholinergic signalling.

BACKGROUND: Airway tuft cells, formerly called brush cells have long been described only morphologically in human airways. More recent RNAseq studies described a chemosensory cell population, which includes tuft cells, by a distinct gene transcription signature. Yet, until which level in the tracheobronchial tree in native human airway epithelium tuft cells occur and if they function as regulators of innate immunity, e.g., by regulating mucociliary clearance, remained largely elusive. METHODS: We performed immunohistochemistry, RT-PCR and immunoblotting analyses for various tuft cell markers to confirm the presence of this cell type in human tracheal samples. Immunohistochemistry was conducted to study the distribution of tuft cells along the intrapulmonary airways in humans. We assessed the influence of bitter substances and the taste transduction pathway on mucociliary clearance in mouse and human tracheal samples by measuring particle transport speed. RESULTS: Tuft cells identified by the expression of their well-established marker POU class 2 homeobox 3 (POU2F3) were present from the trachea to the bronchioles. We identified choline acetyltransferase in POU2F3 expressing cells as well as the transient receptor potential melastatin 5 (TRPM5) channel in a small population of tracheal epithelial cells with morphological appearance of tuft cells. Application of bitter substances, such as denatonium, led to an increase in mucociliary clearance in human tracheal preparations. This was dependent on activation of the TRPM5 channel and involved cholinergic and nitric oxide signalling, indicating a functional role for human tuft cells in the regulation of mucociliary clearance. CONCLUSIONS: We were able to detect tuft cells in the tracheobronchial tree down to the level of the bronchioles. Moreover, taste transduction and cholinergic signalling occur in the same cells and regulate mucociliary clearance. Thus, tuft cells are potentially involved in the regulation of innate immunity in human airways.

PFOS-induced dyslipidemia and impaired cholinergic neurotransmission in developing zebrafish: Insight into its mechanisms.

Perfluorooctane sulfonate (PFOS) is a persistent organic pollutant that has been widely detected in the environment and is known to accumulate in organisms, including humans. The study investigated dose-dependent mortality, hatching rates, malformations, lipid accumulation, lipid metabolism alterations, and impacts on cholinergic neurotransmission. Increasing PFOS concentration led to higher mortality, hindered hatching, and caused concentration-dependent malformations, indicating severe abnormalities in developing zebrafish. The results also demonstrated that PFOS exposure led to a significant increase in total lipids, triglycerides, total cholesterol, and LDL in a concentration-dependent manner, while HDL cholesterol levels were significantly decreased. Additionally, PFOS exposure led to a significant decrease in glucose levels. The study identified TGs, TCHO, and glucose as the most sensitive biomarkers in assessing lipid metabolism alterations. The study also revealed altered expression of genes involved in lipid metabolism, including upregulation of fasn, acaca, and hmgcr and downregulation of ldlr, pparα, and abca1, as well as decreased lipoprotein lipase (LPL) and increased fatty acid synthase (FAS) activity,suggesting an impact on fatty acid synthesis, cholesterol uptake, and lipid transport. Additionally, PFOS exposure led to impaired cholinergic neurotransmission, evidenced by a concentration-dependent inhibition of acetylcholinesterase activity, altered gene expressions related to neural development and function, and reduced Na+/K+-ATPase activity. STRING network analysis highlighted two distinct gene clusters related to lipid metabolism and cholinergic neurotransmission, with potential interactions through the pparα-creb1 pathway. Overall, this study provide important insights into the potential health risks associated with PFOS exposure, including dyslipidemia, cardiovascular disease, impaired glucose metabolism, and neurotoxicity. Further research is needed to fully elucidate the underlying mechanisms and potential long-term effects of PFOS exposure.

Excitatory purinergic and cholinergic expression changed in a partial bladder outlet obstruction-induced overactive bladder rat model.

Overactive bladder (OAB) is a common, long-term symptom complex with a high prevalence in women worldwide. OAB has caused a social burden, and effective treatments are urgently needed. However, the pathogenesis of OAB has yet to be elucidated. Model rats underwent bladder outlet obstruction surgery. In the 2nd, 3rd, and 4th weeks after surgery, metabolic cages were used to detect the 12 h urine volume of rats in the sham and model groups. The urodynamic parameters bladder leak point pressure (BPLL), maximum voiding pressure (MVP), residual volume (RV), maximum bladder capacity (MBC), bladder compliance (BC), voided efficiency (VE), and non-voiding contractions (NVCs) were also detected. Moreover, the contractile responses of isolated detrusor muscles to electrical and carbachol stimulation were examined at the abovementioned time points. At the 4th week after surgery, the bladders of both groups were obtained for hematoxylin-eosin (H&E) and Masson's trichrome staining. Real-time qPCR and Western blot were performed to quantify the expression of choline acetyltransferase (ChAT) and solute carrier family 17 member 9 (SLC17A9). At week 4, compared with the sham group, the 12 h urine volume of PBOO group increased significantly. The BLPP, MVP, VE, MBC, and NVCs increased significantly, and the VE was significantly reduced in 4-week PBOO group. The contractile responses of isolated detrusor muscles to electrical and carbachol stimulation significantly increased in 4-week PBOO group. In the 4-week PBOO group, the bladder wall and the ratio of bladder muscle to collagen within the bladder smooth muscle layer wall were significantly higher than those in the sham group. ChAT and SLC17A9 mRNA and protein expression in the OAB model rats significantly increased. At 4 weeks after PBOO, the OAB model was successfully established. The gene and protein expression levels of ChAT and SLC17A9 increased in the bladder of the OAB model, suggesting that OAB may be related to increased excitatory purinergic and cholinergic expression.

Long-Term Acetylcholinesterase Depletion Alters the Levels of Key Synaptic Proteins while Maintaining Neuronal Markers in the Aging Zebrafish (Danio rerio) Brain.

INTRODUCTION: Interventions targeting cholinergic neurotransmission like acetylcholinesterase (AChE) inhibition distinguish potential mechanisms to delay age-related impairments and attenuate deficits related to neurodegenerative diseases. However, the chronic effects of these interventions are not well described. METHODS: In the current study, global levels of cholinergic, cellular, synaptic, and inflammation-mediating proteins were assessed within the context of aging and chronic reduction of AChE activity. Long-term depletion of AChE activity was induced by using a mutant zebrafish line, and they were compared with the wildtype group at young and old ages. RESULTS: Results demonstrated that AChE activity was lower in both young and old mutants, and this decrease coincided with a reduction in ACh content. Additionally, an overall age-related reduction in AChE activity and the AChE/ACh ratio was observed, and this decline was more prominent in wildtype groups. The levels of an immature neuronal marker were upregulated in mutants, while a glial marker showed an overall reduction. Mutants had preserved levels of inhibitory and presynaptic elements with aging, whereas glutamate receptor subunit levels declined. CONCLUSION: Long-term AChE activity depletion induces synaptic and cellular alterations. These data provide further insights into molecular targets and adaptive responses following the long-term reduction of AChE activity that was also targeted pharmacologically to treat neurodegenerative diseases in human subjects.

Aß1-42 Accumulation Accompanies Changed Expression of Ly6/uPAR Proteins, Dysregulation of the Cholinergic System, and Degeneration of Astrocytes in the Cerebellum of Mouse Model of Early Alzheimer Disease.

Alzheimer disease (AD) is a widespread neurodegenerative disease characterized by the accumulation of oligomeric toxic forms of ß-amyloid (Aß1-42) and dysfunction of the cholinergic system in the different brain regions. However, the exact mechanisms of AD pathogenesis and the role of the nicotinic acetylcholine receptors (nAChRs) in the disease progression remain unclear. Here, we revealed a decreased expression of a number of the Ly6/uPAR proteins targeting nAChRs in the cerebellum of 2xTg-AD mice (model of early AD) in comparison with non-transgenic mice both at mRNA and protein levels. We showed that co-localization of one of them, - neuromodulator Lynx1, with α7-nAChR was diminished in the vicinity of cerebellar astrocytes of 2xTg-AD mice, while Aß1-42 co-localization with this receptor present was increased. Moreover, the expression of anti-inflammatory transcription factor KLF4 regulating transcription of the Ly6/uPAR genes was decreased in the cerebellum of 2xTg-AD mice, while expression of inflammatory cytokine TNF-α was increased. Based on these data together with observed astrocyte degeneration in the cerebellum of 2xTg-AD mice, we suggest the mechanism by which expression of the Ly6/uPAR proteins upon Aß pathology results in dysregulation of the cholinergic system and particularly of α7-nAChR function in the cerebellum. This leads to enhanced neuroinflammation and cerebellar astrocyte degeneration.

Gelatin/polyethylene glycol-loaded magnesium hydroxide nanocomposite to attenuate acetylcholinesterase, neurotoxicity, and activation of GPR55 protein in rat models of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disease marked by mitochondrial dysfunction, amyloid-ß (Aß) aggregation, and neuronal cell loss. G-protein-coupled receptor 55 (GPR55) has been used as a promising target for insulin receptors in diabetes therapy, but GPR55's role in AD is still unidentified. Gelatin (GE) and polyethylene glycol (PEG) polymeric hydrogels are commonly used in the drug delivery system. Therefore, the aim of the present study was the preparation of magnesium hydroxide nanocomposite using Clitoria ternatea (CT) flower extract, GE, and PEG (GE/PEG/Mg(OH)2NCs) by the green precipitation method. The synthesized GE/PEG/Mg(OH)2NCs were used to determine the effect of GPR55 activation of intracerebroventricular administration on streptozotocin (ICV-STC)-induced cholinergic dysfunction, oxidative stress, neuroinflammation, and cognitive deficits. The GE/PEG/Mg(OH)2NCs were administered following bilateral ICV-STC administration (3 mg/kg) in experimental rats. Neurobehavioral assessments were performed using a Morris water maze (MWM) and a passive avoidance test (PA). Cholinergic and antioxidant activity, oxidative stress, and mitochondrial complex activity were estimated in the cortex and hippocampus through biochemical analysis. Inflammatory markers (TNF-α, IL-6, and IL-1ß) were determined using the ELISA method. Our study results demonstrated that the GE/PEG/Mg(OH)2NCs treatment significantly improved spatial and non-spatial memory functions in behavioral studies. Moreover, the treatment with GE/PEG/Mg(OH)2NCs group significantly attenuated cholinergic dysfunction, oxidative stress, and inflammatory markers, and also highly improved anti-oxidant activity (GSH, SOD, CAT, and GPx) in the cortex and hippocampus regions. The western blot results suggest the activation of the GPR55 protein expression through GE/PEG/Mg(OH)2NCs. The histopathological studies showed clear cytoplasm and healthy neurons, effectively promoting neuronal activity. Furthermore, the molecular docking results demonstrated the binding affinity and potential interactions of the compounds with the AChE enzyme. In conclusion, the GE/PEG/Mg(OH)2NCs treated groups showed reduced neurotoxicity and have the potential as a therapeutic agent to effectively target AD.