Choline Acetyltransferase Protein Research Articles

Repetitive Mild but Not Single Moderate Brain Trauma Is Associated with TAR DNA-Binding Protein 43 Mislocalization and Glial Activation in the Mouse Spinal Cord.

Background/Objectives: Traumatic brain injury (TBI) occurs after a sudden mechanical force to the skull and represents a significant public health problem. Initial brain trauma triggers secondary pathophysiological processes that induce structural and functional impairment of the central nervous system, even in the regions distant to the lesion site. Later in life, these changes can be manifested as neurodegenerative sequalae that commonly involve proteinopathies, such as transactive DNA-binding protein 43 (TDP-43). The progression of pathophysiological changes to the spinal cord motor neurons has been detected after repetitive TBI, while such changes have been less investigated after single TBI. Methods: Single TBI was applied over the left parietal cortex of mice by using the lateral fluid percussion injury apparatus and a separate cohort of animals received repetitive mild TBI by weight drop apparatus, with two mild injuries daily, for five days in a row. Mice were sacrificed after single moderate or last mild TBI and their spinal cords were prepared for the analyses. For both types of injury, sham-injured mice were used as a control group. Results: Here, we found an early formation of toxic phosphorylated TDP-43 species on the 3rdday post-injury which, together with TDP-43 cytoplasmic translocation, remained present in the subacute period of 14 days after repetitive mild but not single moderate TBI. During the subacute period following a repetitive brain trauma, we found an increased choline acetyltransferase protein expression and significant microgliosis in the cervical part of the spinal cord, which was not detected after single TBI. Astrogliosis presented similarly after both experimental procedures. Conclusions: This study demonstrates the differences in the spinal cord TDP-43 pathology and inflammation, depending on the brain trauma type, and may contribute to the development of targeted therapeutic strategies.

Amphetamine Injection into the Nucleus Accumbens and Electrical Stimulation of the Ventral Tegmental Area in Rats After Novelty Test-Behavioral and Neurochemical Correlates.

Amphetamine abuse is a global health epidemic that is difficult to treat due to individual differences in response to environmental factors, including stress reactivity and anxiety levels, as well as individual neuronal differences, which may result in increased/decreased vulnerability to addiction. In the present study, we investigated whether the Wistar rats behavioral traits of high (HR) and low (LR) locomotor activity to novelty influence motivational behavior (induced feeding model; iFR by electrical stimulation of the ventral tegmental area; Es-VTA) supported by amphetamine injection into the nucleus accumbens shell (AcbSh) (HRAmph, n = 5; LRAmph, n = 5). A correlation was found between the novelty test's locomotor activity score and the frequency threshold percentage change (p < 0.001, Rs = -0.867). In HRAmph, there was a shortening (-24.16%), while in LRAmph, there was a lengthening (+51.84%) of iFR latency. Immunofluorescence studies showed differential neuronal density (activity of tyrosine hydroxylase, choline acetyltransferase, and cFos protein) in the selected brain structures in HRAmph and LRAmph animals as well as in comparison to a control group (HRACSF, n = 5; LRACSF, n = 5). These results contribute to expanding the state of knowledge of the behavioral and neuronal propensity to take drug abuse.

Micellar Choline-Acetyltransferase Complexes Exhibit Ultra-Boosted Catalytic Rate for Acetylcholine Synthesis-Mechanistic Insights for Development of Acetylcholine-Enhancing Micellar Nanotherapeutics.

Choline-acetyltransferase (ChAT) is the key cholinergic enzyme responsible for the biosynthesis of acetylcholine (ACh), a crucial signaling molecule with both canonical neurotransmitter function and auto- and paracrine signaling activity in non-neuronal cells, such as lymphocytes and astroglia. Cholinergic dysfunction is linked to both neurodegenerative and inflammatory diseases. In this study, we investigated a serendipitous observation, namely that the catalytic rate of human recombinant ChAT (rhChAT) protein greatly differed in buffered solution in the presence and absence of Triton X-100 (TX100). At a single concentration of 0.05% (v/v), TX100 boosted the specific activity of rhChAT by 4-fold. Dose-response analysis within a TX100 concentration range of 0.8% to 0.008% (accounting for 13.7 mM to 0.013 mM) resulted in an S-shaped response curve, indicative of an over 10-fold boost in the catalytic rate of rhChAT. This dramatic boost was unlikely due to a mere structural stabilization since it remained even after the addition of 1.0 mg/mL gelatin to the ChAT solution as a protein stabilizer. Furthermore, we found that the catalytic function of the ACh-degrading enzyme, AChE, was unaffected by TX100, underscoring the specificity of the effect for ChAT. Examination of the dose-response curve in relation to the critical micelle concentration (CMC) of TX100 revealed that a boost in ChAT activity occurred when the TX100 concentration passed its CMC, indicating that formation of micelle-ChAT complexes was crucial. We challenged this hypothesis by repeating the experiment on Tween 20 (TW20), another non-ionic surfactant with ~3-fold lower CMC compared to TX100 (0.06 vs. 0.2 mM). The analysis confirmed that micelle formation is crucial for ultra-boosting the activity of ChAT. In silico molecular dynamic simulation supported the notion of ChAT-micelle complex formation. We hypothesize that TX100 or TW20 micelles, by mimicking cell-membrane microenvironments, facilitate ChAT in accessing its full catalytic potential by fine-tuning its structural stabilization and/or enhancing its substrate accessibility. These insights are expected to facilitate research toward the development of new cholinergic-enhancing therapeutics through the formulation of micelle-embedded ChAT nanoparticles.

Thyroid Hormone Neuroprotection Against Perfluorooctane Sulfonic Acid Cholinergic and Glutamatergic Disruption and Neurodegeneration Induction.

Background: Perfluorooctane sulfonic acid (PFOS), a widely used industrial chemical, was reported to induce memory and learning process dysfunction. Some studies tried to reveal the mechanisms that mediate these effects, but how they are produced is still unknown. Basal forebrain cholinergic neurons (BFCN) maintain cognitive function and their selective neurodegeneration induces cognitive decline, as observed in Alzheimer's disease. PFOS was reported to disrupt cholinergic and glutamatergic transmissions and thyroid hormone action, which regulate cognitive processes and maintain BFCN viability. Objective/Methods: To evaluate PFOS neurodegenerative effects on BFCN and the mechanisms that mediate them, SN56 cells (a neuroblastoma cholinergic cell line from the basal forebrain) were treated with PFOS (0.1 µM to 40 µM) with or without thyroxine (T3; 15 nM), MK-801 (20 µM) or acetylcholine (ACh; 10 µM). Results: In the present study, we found that PFOS treatment (1 or 14 days) decreased thyroid receptor α (TRα) activity by decreasing its protein levels and increased T3 metabolism through increased deiodinase 3 (D3) levels. Further, we observed that PFOS treatment disrupted cholinergic transmission by decreasing ACh content through decreased choline acetyltransferase (ChAT) activity and protein levels and through decreasing muscarinic receptor 1 (M1R) binding and protein levels. PFOS also disrupted glutamatergic transmission by decreasing glutamate content through increased glutaminase activity and protein levels and through decreasing N-methyl-D-aspartate receptor subunit 1 (NMDAR1); effects mediated through M1R disruption. All these effects were mediated through decreased T3 activity and T3 supplementation partially restored to the normal state. Conclusions: These findings may assist in understanding how PFOS induces neurodegeneration, and the mechanisms involved, especially in BFCN, to explain the process that could lead to cognitive dysfunction and provide new therapeutic tools to treat and prevent its neurotoxic effects.

Chronic inflammation with microglia senescence at basal forebrain: impact on cholinergic deficit in Alzheimer's brain haemodynamics.

Cholinergic innervation in the brain is involved in modulating neurovascular function including cerebral blood flow haemodynamics in response to neuronal activity. Cholinergic deficit is associated with pathophysiology in Alzheimer's disease, albeit the aetiology remains to be clarified. In the current study, neocortex cerebral blood flow response to acetylcholine was evaluated by Laser-Doppler Flowmetry (LDF) in 3xTgAD Alzheimer's disease model) and wild-type mice of two age groups. The peak of cerebral blood flow to acetylcholine (i.v.) from baseline levels (% ΔrCBF) was higher in young 3xTgAD versus in wild-type mice (48.35; 95% CI:27.03-69.67 versus 22.70; CI:15.5-29.91, P < 0.05); this was reversed in old 3xTgAD mice (21.44; CI:2.52-40.35 versus 23.25; CI:23.25-39). Choline acetyltransferase protein was reduced in neocortex, while cerebrovascular reactivity to acetylcholine was preserved in young 3×TgAD mice. This suggests endogenous acetylcholine deficit and possible cholinergic denervation from selected cholinergic nuclei within the basal forebrain. The early deposition of tauopathy moieties (mutant hTau and pTau181) and its coincidence in cholinergic cell clusters (occasionaly), were observed at the basal forebrain of 3xTgAD mice including substantia innominate, nucleus Basalis of Meynert and nucleus of horizontal limb diagonal band of Broca. A prominent feature was microglia interacting tauopathy and demonstrated a variety of morphology changes particularly when located in proximity to tauopathy. The microglia ramified phenotype was reduced as evaluated by the ramification index and Fractal analysis. Increased microglia senescence, identified as SASP (senescence-associated secretory phenotype), was colocalization with p16Ink4ɑ, a marker of irreversible cell-cycle arrest in old 3xTgAD versus wild-type mice (P = 0.001). The p16Ink4ɑ was also observed in neuronal cells bearing tauopathy within the basal forebrain of 3xTgAD mice. TNF-ɑ, the pro-inflammatory cytokine elevated persistently in microglia (Pearson's correlation coefficient = 0.62) and the loss of cholinergic cells in vulnerable basal forebrain environment, was indicated by image analysis in 3xTgAD mice, which linked to the cholinergic deficits in neocortex rCBF haemodynamics. Our study revealed the early change of CBF haemodynamics to acetylcholine in 3xTgAD model. As a major effector of brain innate immune activation, microglia SASP with age-related disease progression is indicative of immune cell senescence, which contributes to chronic inflammation and cholinergic deficits at the basal forebrain. Targeting neuroinflammation and senescence may mitigate cholinergic pathophysiology in Alzheimer's disease.

Scutellaria Baicalensis Georgi Stem and Leaf Flavonoids Ameliorate the Learning and Memory Impairment in Rats Induced by Okadaic Acid.

The objective of this study is to explore the impact and underlying mechanism of Scutellaria baicalensis Georgi stem and leaf flavonoids (SSFs) on cognitive impairment caused by intracerebroventricular injection of okadaic acid (OA) in rats. An experimental model of Alzheimer's disease (AD) was induced in rats by intracerebroventricular injection of OA, resulting in memory impairment. The Morris water maze test was employed to confirm the successful establishment of the memory impairment model. The rats that exhibited significant memory impairment were randomly divided into different groups, including a model group, three SSFs dose groups (25, 50, and 100 mg/kg), and a positive control group treated with Ginkgo biloba tablets (GLT) at a dose of 200 mg/kg. To evaluate the learning and memory abilities of the rats, the Morris water maze test was conducted. Hematoxylin-eosin (HE) staining was used to observe any morphological changes in neurons. Immunohistochemistry (IHC) was performed to measure the expression of choline acetyltransferase (ChAT) protein. Western blotting (WB) was utilized to assess the phosphorylation levels of tau protein at Ser262 and Ser396. The activities of inducible nitric oxide synthase (iNOS) and constitutive nitric oxide synthase (cNOS) were quantified using ultraviolet spectrophotometry. The levels of inflammatory factors, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), were measured using ELISA. In rats, the administration of OA via intracerebroventricular injection resulted in cognitive impairment, neuropathological changes, and alterations in protein expression and activity levels. Specifically, the protein expression of ChAT was significantly reduced (P<0.01), while the phosphorylation levels of tau protein at Ser262 and Ser396 were significantly increased (P<0.01). Moreover, iNOS activity in the hippocampus and cerebral cortex exhibited a significant increase (P<0.01), whereas cNOS activity showed a decrease (P<0.05). Furthermore, the levels of IL-1β and TNF-α in the cerebral cortex were elevated (P<0.01), while the level of IL-6 was decreased (P<0.05). The administration of three doses of SSFs and GLT to rats exhibited varying degrees of improvement in the aforementioned pathological alterations induced by OA. SSFs demonstrated the ability to enhance cognitive function and mitigate memory deficits in rats following intracerebroventricular injection of OA. This beneficial effect may be attributed to the modulation of ChAT protein expression, tau hyperphosphorylation, NOS activity, and inflammatory cytokine levels by SSFs.

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.

Regulation of enteric nervous system via sacral nerve stimulation in opioid-induced constipated rats.

Sacral nerve stimulation (SNS) has been employed for treating constipation. However, its mechanisms involving enteric nervous system (ENS) and motility are largely unknown. In this study, we investigated the possible ENS involvement of SNS in treating Loperamide-induced constipation in rats. Experiment-1 was designed to study the effects of acute SNS on whole colon transit time (CTT). In experiment-2, we induced constipation by Loperamide and then applied daily SNS or sham-SNS for 1 week. Choline acetyltransferase (ChAT), nitric oxide synthase (nNOS), and PGP9.5 in colon tissue were examined at the end of the study. Moreover, survival factors such as phosphorylated AKT (p-AKT) and Glial cell-derived neurotrophic factor (GDNF) were measures by immunohistochemistry (IHC) and western blot (WB). (1) SNS with one set of parameters shortened CTT starting at 90 min after phenol red administration (p < 0.05). (2) While Loperamide induced slow transit constipation with a significant reduction in fecal pellet number and feces wet weight, daily SNS for a week resolved constipation. (3) Moreover, SNS was able to shorten whole gut transit time comparing to sham-SNS (p = 0.01). (4) Loperamide reduced the number of PGP9.5 and ChAT positive cells, and downregulated ChAT protein expression and upregulated nNOS protein expression, whereas these detrimental effects were significantly reversed by SNS. (5) Furthermore, SNS increased expressions of both GDNF and p-AKT in colon tissue. (6) Vagal activity was reduced following Loperamide (p < 0.01); yet SNS normalized vagal activity. SNS with appropriate parameters improves opioid-induced constipation and reversed the detrimental effects of Loperamide on enteric neurons possibly via the GDNF-PI3K/Akt pathway.GRAPHICAL ABSTRACT.

Effect of electroacupuncture at "Zusanli" (ST36) and "Tianshu" (ST25) on intestinal function and autonomic nerve balance in rats with irritable bowel syndrome

To study whether electroacupuncture (EA) of "Zusanli" (ST36) combined with "Tianshu" (ST25) has a synergistic effect in regulating the colonic function and autonomic nerve balance in rats with irritable bowel syndrome (IBS). Male Wistar rats were randomly divided into control, model, EA-ST36, and EA-ST36+ST25 groups, with 14 rats in each group. The IBS model was established by using water avoidance stress method. The visceral hypersensitivity was measured using the abdominal wall retraction reflex (AWR). The rectus abdominis electromyogram (EMG), intestinal electrical activity, and electrocardiogram (ECG) were recorded using a PowerLab data acquisition and analysis system. The contents of serum cAMP and cGMP were determined by ELISA, the expression levels of colonic tyrosine hydroxylase (TH) and choline acetyl-transferase (ChAT) proteins were determined by immunofluorescence staining and Western blot, respectively. Compared with the control group, the model group had an evident increase in the levels of AWR, LF, LF/HF, ChAT protein expression, cAMP and cGMP contents and cAMP/cGMP ratio (P<0.001, P<0.05), and a marked decrease in the levels of HF, frequency of slow waves of intestinal EMG, visceral pain threshold (PT), immunoactivity and expression of TH protein (P<0.05, P<0.001). In contrast to the model group, the levels of AWR, LF, LF/HF, ChAT protein expression and immunoactivity, cAMP and cGMP contents and ratio of cAMP/cGMP were significantly reduced (P<0.001, P<0.05, P<0.01), whereas the levels of frequency of slow waves of intestinal EMG, PT, and the immunoactivity and expression of TH were considerably increased (P<0.001, P<0.05) in both EA-ST36 and EA-ST36+ST25 groups. EA of both ST36 and ST36+ST25 can relieve visceral pain, and reduce sympathetic activity to improve autonomic nerve balance, but without apparent synergistic effect between EA-ST36 and EA-ST25 in rats with IBS.

A novel transgenic mouse model expressing primate-specific nuclear choline acetyltransferase: insights into potential cholinergic vulnerability

The acetylcholine (ACh) synthesizing enzyme choline acetyltransferase (ChAT) is an important cholinergic neuronal marker whose levels and/or activity are reduced in physiological and pathological aging. One isoform of ChAT, 82-kDa ChAT, is expressed only in primates and found primarily in nuclei of cholinergic neurons in younger individuals, but this protein becomes mostly cytoplasmic with increasing age and in Alzheimer’s disease (AD). Previous studies suggest that 82-kDa ChAT may be involved in regulating gene expression during cellular stress. Since it is not expressed in rodents, we developed a transgenic mouse model that expresses human 82-kDa ChAT under the control of an Nkx2.1 driver. Behavioral and biochemical assays were used to phenotype this novel transgenic model and elucidate the impact of 82-kDa ChAT expression. The 82-kDa ChAT transcript and protein were expressed predominantly in basal forebrain neurons and subcellular distribution of the protein recapitulated the age-related pattern found previously in human necropsy brains. Older 82-kDa ChAT-expressing mice presented with better age-related memory and inflammatory profiles. In summary, we established a novel transgenic mouse expressing 82-kDa ChAT that is valuable for studying the role of this primate-specific cholinergic enzyme in pathologies associated with cholinergic neuron vulnerability and dysfunction.

ODP228 Obesity Induced a Down-Regulation of nNOS in Intrapancreatic Nervous System

Abstract Objective Obesity has become a serious global public health problem. Obesity is frequently preceding and the most important risk factor of type 2 diabetes. Pancreas regulates glucose metabolism and glucose homeostasis, and abnormality of pancreas play an important role in insulin resistance and diabetes. Intrapancreatic nerve innervation modulates insulin release and is tightly linked to the onset and development of diabetes. However, it is not known whether the activity of the intrapancreatic nervous system has been altered before the onset of diabetes. In this study, we examined the intrapancreatic nervous system in rats with obesity looking for the changes induced by obesity. Methods Obese Sprague-Dawley rats were developed with feeding high fat diet for 10 weeks. Using Immunofluorescence method, choline acetyl transferase (ChAT) and nNOS positive neurons in intrapancreatic ganglia were counted. ChAT and nNOS expression in pancreas were also measured with western blot method. Results (1) There was no significant difference in fasting blood glucose between obese rats and normal rats, but oral glucose tolerance test showed that the oral glucose tolerance was impaired in obese rats. (2) In rats fed with normal diet, 80.2%±1.5 neurons in intrapancreatic ganglia were ChAT positive, and in the obese rats, 79.3%±1.8 neurons were ChAT positive. There was no significant difference between the two groups (p&amp;gt;0. 05, n=5). In intrapancreatic ganglia of obese rats, 40%±1.4 neurons were nNOS positive, which was significantly lower than that (60.5%±1.2) of rats fed with normal diet (p&amp;lt;0. 01,n=5). (3) In pancreatic tissue, the relative expression level of ChAT protein was 1. 02±0. 03 in rats fed with normal diet and 0.98±0. 05 in obese rats (p&amp;gt;0. 05, n=5). The relative expression level of nNOS protein was 1.70±0. 08 in normal rats and 1.10±0. 05 in obese rats (p&amp;gt;0. 01, n=5). Conclusion The cholinergic nerves in intrapancreatic nervous system was not altered in obese rats, however, nNOS nerves was down-regulated in obese rats. The down-regulation of nNOS would induce a hyperactivity in intrapancreatic nervous system and this would cause an enhancement of insulin release, which has been observed in obese individuals. Presentation: No date and time listed

Human Pulmonary Vein Myocardial Sleeve Autonomic Neural Density and Cardiovascular Mortality.

Myocardial sleeves around pulmonary veins (PVs) are highly innervated structures with heterogeneous morphological and electrophysiological characteristics. Autonomic nerve dysfunction in the myocardium may be associated with an increased risk of cardiovascular morbidity and mortality. This article studied autonomic neural remodeling in myocardial sleeves around PVs and atrial-PV ostia with immunohistochemical and morphometric methods with clinicopathological correlations. PVs were collected from 37 and atrial-PV ostia from 17 human autopsy hearts. Immunohistochemical analysis was performed using antibodies against tyrosine hydroxylase (TH), choline acetyltransferase (CHAT), and growth-associated protein 43 (GAP43). In the PV cohort, subjects with immediate cardiovascular cause of death had significantly decreased sympathetic nerve density in fibro-fatty tissue vs those with non-cardiovascular cause of death (1624.53 vs 2522.05 µm2/mm2, p=0.038). In the atrial-PV ostia cohort, parasympathetic nerve density in myocardial sleeves was significantly increased in subjects with underlying cardiovascular cause of death (19.48 µm2/mm2) than subjects with underlying non-cardiovascular cause of death with no parasympathetic nerves detected (p=0.034). Neural growth regionally varied in sympathetic nerves and was present in most of the parasympathetic nerves. Heterogeneous autonomic nerve distribution and growth around PVs and atrial-PV ostia might play a role in cardiovascular morbidity and mortality. No association in nerve density was found with atrial fibrillation.

Study of lead-induced neurotoxicity in cholinergic cells differentiated from bone marrow-derived mesenchymal stem cells.

The developing brain is susceptible to the neurotoxic effects of lead. Exposure to lead has main effects on the cholinergic system and causes reduction of cholinergic neuron function during brain development. Disruption of the cholinergic system by chemicals, which play important roles during brain development, causes of neurodevelopmental toxicity. Differentiation of stem cells to neural cells is recently considered a promising tool for neurodevelopmental toxicity studies. This study evaluated the toxicity of lead acetate exposure during the differentiation of bone marrow-derived mesenchyme stem cells (bone marrow stem cells, BMSCs) to CCholinergic neurons. Following institutional animal care review board approval, BMSCs were obtained from adult rats. The differentiating protocol included two stages that were pre-induction with β-mercaptoethanol (BME) for 24h and differentiation to cholinergic neurons with nerve growth factor (NGF) over 5days. The cells were exposed to different lead acetate concentrations (0.1-100μm) during three stages, including undifferentiated, pre-induction, and neuronal differentiation stages; cell viability was measured by MTT assay. Lead exposure (0.01-100μg/ml) had no cytotoxic effect on BMSCs but could significantly reduce cell viability at 50 and 100μm concentrations during pre-induction and neuronal differentiation stages. MAP2 and choline acetyltransferase (ChAT) protein expression were investigated by immunocytochemistry. Although cells treated with 100μm lead concentration expressed MAP2 protein in the differentiation stages, they had no neuronal cell morphology. The ChAT expression was negative in cells treated with lead. The present study showed that differentiated neuronal BMSCs are sensitive to lead toxicity during differentiation, and it is suggested that these cells be used to study neurodevelopmental toxicity.

Identification of cholinergic cells with chemosensory traits in the porcine uterus.

Chemosensory cells are specialized epithelial cells that act as sentinels near body entry sites. The majority of these cells express a cholinergic phenotype and utilize the taste signaling system to monitor the mucosal environment for potentially harmful substances, triggering protective reflexes. We report the identification of cells with a putative chemosensory role in the uterus. Presumptive chemosensory cells were immunoreactive to key components of the taste transduction, including the transient receptor potential channel M5 (TRPM5) and the phospholipase Cβ2 (PLCB2). These cells localized to endometrial glandular and luminal epithelia, while absent from myometrium and perimetrium. Double immunofluorescence revealed co-expression of chemosensory cell markers with the acetylcholine (ACh) synthesizing enzyme, choline acetyltransferase (ChAT). Further, we investigated the regional distribution and expression of chemosensory cells at different stages of the estrous cycle. Uteri were collected postmortem from gilts and stages of the ovarian cycle were determined macroscopically. The uteri were classified into three groups: prepubertal (PB), follicular (FOL), or luteal (LUT). The number of ChAT-immunoreactive cells was increased in the luminal epithelium in the caudal compartment compared to the cranial region of the uterine horn, and at the LUT compared to PB and FOL stages. An increase in ChAT protein abundance in LUT uterine homogenates was noted, although not followed by an increase in ACh content. In summary, our study has identified a hitherto unrecognized cholinergic cell in the uterus that has chemosensory traits and may be involved in a multitude of biological processes.

Selenium enhances the expression of miR-9, miR-124 and miR-29a during neural differentiation of bone marrow mesenchymal stem cells

BackgroundSelenium (Se) is a trace element that plays important role in antioxidant defense in the brain. Sodium selenite (Na2SeO3) is an inorganic salt of Se which has an antioxidant function. In the present study, we investigated the effect of Sodium selenite on the expression of important neuronal microRNAs during neural differentiation of bone marrow-derived stem cells (BMSCs). MethodsMesenchymal stem cells were collected from rat bone marrow and cultured in the Dulbecco's Modified Eagle Medium (DMEM) medium. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay was conducted to determine the toxicity of Na2SeO3. For neural induction, BMSCs were divided into control, Na2SeO3 containing (10 ng/mL) and Na2SeO3 free groups and cultured in DMEM medium supplemented with Isobutyl-l-methylxanthine (IBMX), Fibroblast growth factor 2 (FGF2), B27, Retinoic acid, and brain derived neurotrophic factor (BDNF) for 14 days. At the end of the differentiation, immunostaining against Microtubule associated protein 2 (Map-2) and Choline acetyltransferase (ChAT) proteins was performed. Also, the total RNA is extracted from control and neural differentiated cells using a special kit, and the expression of miR-9, miR-124, and miR-29a was analyzed using real-time polymerase chain reaction (RT-PCR). ResultsIncreasing Na2SeO3 concentrations had increasing toxicity; therefore, the concentration of 10 ng/mL was used as a supplement during neural differentiation. Examination of the expression of Map-2 and ChAT proteins showed that Na2SeO3 increased the expression of them and consequently the neuronal differentiation of BMSCs. Na2SeO3 also significantly increased the expression of miR-9, miR-124, and miR-29a in BMSCs undergoing neuronal differentiation. ConclusionsOur results suggest that the protective effect of selenium on neural differentiation of stem cells may be mediated through neuron specific microRNAs. This result further highlights the importance of selenium supplementation in preventing neuronal diseases.

CSF and Plasma Cholinergic Markers in Patients With Cognitive Impairment.

IntroductionAlzheimer’s disease (AD) is the most prevalent form of dementia with symptoms of deteriorating cognitive functions and memory loss, partially as a result of a decrease in cholinergic neurotransmission. The disease is incurable and treatment with cholinesterase inhibitors (ChEIs) is symptomatic. Choline acetyltransferase (ChAT), the enzyme that synthesizes acetylcholine (ACh), has been proven recently to be present in both cerebrospinal fluid (CSF) and plasma. As ChAT plays a role in regulating the extracellular ACh levels, it may have an impact on prognosis and cognitive performance in AD patients.ObjectivesTo measure ChAT activity and its protein concentration in CSF and plasma from patients with AD, mild cognitive impairment (MCI), or Subjective cognitive impairment (SCI).MethodsPlasma and CSF samples were obtained from 21 AD, 32 MCI, and 30 SCI patients. The activity and protein levels of ChAT and acetylcholinesterase (AChE), the enzyme catalyzing the hydrolysis of ACh, were analyzed using an integrated activity and protein concentration ELISA-like assay. A Cholinergic Index was calculated as the ratio of ChAT to AChE activities in CSF. The data were analyzed in relation to dementia biomarkers and cognitive performance of the patients.ResultsThe CSF ChAT activity was significantly higher (55–67%) in MCI patients compared to AD and SCI cases. The CSF Cholinergic Index was 41 and 22% lower in AD patients than in MCI and SCI subjects, respectively. This index correlated positively with the Aβ42/p-tau ratio in CSF in SCI but negatively with that in AD and MCI. The ChAT activity and protein levels in plasma exhibited significant differences with the pattern of AD>>MCI>SCI.ConclusionThis is the first study investigating soluble levels of the key cholinergic enzyme, ChAT, in both plasma and CSF of individuals at different clinical stages of dementia. Although further validation is needed, the overall pattern of the results suggests that in the continuum of AD, the cholinergic signaling exhibits an inverse U-shape dynamic of changes in the brain that greatly differs from the changes observed in the plasma compartment.

Analysis of Caenorhabditis elegans acetylcholine synthesis mutants reveals a temperature-sensitive requirement for cholinergic neuromuscular function.

In Caenorhabditis elegans, the cha-1 gene encodes choline acetyltransferase (ChAT), the enzyme that synthesizes the neurotransmitter acetylcholine. We have analyzed a large number of cha-1 hypomorphic mutants, most of which are missense alleles. Some homozygous cha-1 mutants have approximately normal ChAT immunoreactivity; many other alleles lead to consistent reductions in synaptic immunostaining, although the residual protein appears to be stable. Regardless of protein levels, neuromuscular function of almost all mutants is temperature-sensitive, i.e., neuromuscular function is worse at 25° than at 14°. We show that the temperature effects are not related to acetylcholine release, but specifically to alterations in acetylcholine synthesis. This is not a temperature-dependent developmental phenotype, because animals raised at 20° to young adulthood and then shifted for 2 h to either 14° or 25° had swimming and pharyngeal pumping rates similar to animals grown and assayed at either 14° or 25°, respectively. We also show that the temperature-sensitive phenotypes are not limited to missense alleles; rather, they are a property of most or all severe cha-1 hypomorphs. We suggest that our data are consistent with a model of ChAT protein physically, but not covalently, associated with synaptic vesicles; and there is a temperature-dependent equilibrium between vesicle-associated and cytoplasmic (i.e., soluble) ChAT. Presumably, in severe cha-1 hypomorphs, increasing the temperature would promote dissociation of some of the mutant ChAT protein from synaptic vesicles, thus removing the site of acetylcholine synthesis (ChAT) from the site of vesicular acetylcholine transport. This, in turn, would decrease the rate and extent of vesicle-filling, thus increasing the severity of the behavioral deficits.

Activation of cholinergic system partially rescues olfactory dysfunction-induced learning and memory deficit in mice

Deficits in olfaction are associated with neurodegenerative disorders such as Alzheimer’s disease. A recent study reported that intranasal zinc sulfate (ZnSO4)-treated mice show olfaction and memory deficits. However, it remains unknown whether olfaction deficit-induced learning and memory impairment is associated with the cholinergic system in the brain. In this study, we evaluated olfactory function by the buried food find test, and learning and memory function by the Y-maze and passive avoidance tests in ZnSO4-treated mice. The expression of choline acetyltransferase (ChAT) protein in the olfactory bulb (OB), prefrontal cortex, hippocampus, and amygdala was assessed by western blotting. Moreover, we observed the effect of the acetylcholinesterase inhibitor physostigmine on ZnSO4-induced learning and memory deficits. We found that intranasal ZnSO4-treated mice exhibited olfactory dysfunction, while this change was recovered on day 14 after treatment. Both short-term and long-term learning and memory were impaired on days 4 and 7 after treatment with ZnSO4, whereas the former, but not the latter, was recovered on day 14 after treatment. A significant correlation was observed between olfactory function and short-term memory, but not long-term memory. Treatment with ZnSO4 decreased the ChAT level in the OB on day 4, and increased and decreased the ChAT levels in the OB and hippocampus on day 7, respectively. Physostigmine improved the ZnSO4-induced deficit in short-term, but not long-term, memory. Taken together, the present results suggest that short-term memory may be closely associated with olfactory function via the cholinergic system.

Alteration of the cholinergic system and motor deficits in cholinergic neuron-specific Dyt1 knockout mice

Dystonia is a neurological movement disorder characterized by sustained or intermittent muscle contractions, repetitive movement, and sometimes abnormal postures. DYT1 dystonia is one of the most common genetic dystonias, and most patients carry heterozygous DYT1 ∆GAG mutations causing a loss of a glutamic acid of the protein torsinA. Patients can be treated with anticholinergics, such as trihexyphenidyl, suggesting an abnormal cholinergic state. Early work on the cell-autonomous effects of Dyt1 deletion with ChI-specific Dyt1 conditional knockout mice (Dyt1 Ch1KO) revealed abnormal electrophysiological responses of striatal ChIs to muscarine and quinpirole, motor deficits, and no changes in the number or size of the ChIs. However, the Chat-cre line that was used to derive Dyt1 Ch1KO mice contained a neomycin cassette and was reported to have ectopic cre-mediated recombination. In this study, we generated a Dyt1 Ch2KO mouse line by removing the neomycin cassette in Dyt1 Ch1KO mice. The Dyt1 Ch2KO mice showed abnormal paw clenching behavior, motor coordination and balance deficits, impaired motor learning, reduced striatal choline acetyltransferase protein level, and a reduced number of striatal ChIs. Furthermore, the mutant striatal ChIs had a normal muscarinic inhibitory function, impaired quinpirole-mediated inhibition, and altered current density. Our findings demonstrate a cell-autonomous effect of Dyt1 deletion on the striatal ChIs and a critical role for the striatal ChIs and corticostriatal pathway in the pathogenesis of DYT1 dystonia.

Plasma Rich in Growth Factors Enhances Cell Survival after in Situ Retinal Degeneration

Purpose: The purpose of this study was to examine the effect of plasma rich in growth factors (PRGFs) under blue light conditions in an in vivo model of retinal degeneration. Methods: Male Wistar rats were exposed to dark/blue light conditions for 9 days. On day 7, right eyes were injected with saline and left eyes with PRGF. Electroretinography (ERG) and intraocular pressure (IoP) measurements were performed before and after the experiment. After sacrifice, retinal samples were collected. Hematoxylin and eosin staining was performed to analyze the structure of retinal sections. Immunofluorescence for brain-specific homeobox/POU domain protein 3A (Brn3a), choline acetyltransferase (ChAT), rhodopsin, heme oxygenase-1 (HO-1), and glial fibrillary acidic protein (GFAP) was performed to study the retinal conditions. Results: Retinal signaling measured by ERG was reduced by blue light and recovered with PRGF; however, IoP measurements did not show significant differences among treatments. Blue light reduced the expression for Brn3a, ChAT, and rhodopsin. Treatment with PRGF showed a recovery in their expressions. HO-1 and GFAP results showed that blue light increased their expression but the use of PRGF reduced the effect of light. Conclusions: Blue light causes retinal degeneration. PRGF mitigated the injury, restoring the functionality of these cells and maintaining the tissue integrity.