Protein Expression Of ChAT Research Articles

RNAi and chemogenetic reporter co-regulation in primate striatal interneurons

Using genetic tools to study the functional roles of molecularly specified neuronal populations in the primate brain is challenging, primarily because of specificity and verification of virus-mediated targeting. Here, we report a lentivirus-based system that helps improve specificity and verification by (a) targeting a selected molecular mechanism, (b) in vivo reporting of expression, and (c) allowing the option to independently silence all regional neural activity. Specifically, we modulate cholinergic signaling of striatal interneurons by shRNAmir and pair it with hM4Di_CFP, a chemogenetic receptor that can function as an in vivo and in situ reporter. Quantitative analyses by visual and deep-learning assisted methods show an inverse linear relation between hM4Di_CFP and ChAT protein expression for several shRNAmir constructs. This approach successfully applies shRNAmir to modulating gene expression in the primate brain and shows that hM4Di_CFP can act as a readout for this modulation.

Effects of Resveratrol on the Mechanisms of Antioxidants and Estrogen in Alzheimer's Disease.

Objective To observe the effects of resveratrol (Res) on the antioxidative function and estrogen level in an Alzheimer's disease (AD) mouse model. Methods First, we examined the effects of Res on an AD mice model. SAMP8 mice were selected as the model, and normal-aging SAMR1 mice were used as the control group. The model mice were randomly divided into three groups: a model group, high-dose Res group (40mg/kg, intraperitoneal (ip)), and low-dose Res group (20mg/kg, ip). After receiving medication for 15 days, the mice were subjected to the water maze test to assess their spatial discrimination. The spectrophotometric method was used to detect the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) as well as the malondialdehyde (MDA) content. Quantitative PCR (q-PCR) was used to detect SOD, GSH-Px, CAT, and heme oxygenase-1 (HO-1) mRNA level changes. Western blot analysis detected HO-1 and Nrf2 protein expression. Second, we researched the effect of Res on the estrogen level in the SAMP8 model mice. The model mice were randomly divided into four groups: a model group, estrogen replacement group (0.28 mg/kg, intramuscular (im), estradiol benzoate), high-dose Res group (5 mg/kg, im), and low-dose Res group (2.5 mg/kg, im). The mice were injected, once every three days, for 5 weeks. Q-PCR was used to detect brain tissue mRNA expression changes. Western blot analysis detected ERα, ERβ, and ChAT protein expression. An enzyme-linked immunosorbent assay (ELISA) kit was used to detect the expression of E2 and amyloid β protein (Aβ) in brain tissue. Results Compared with the control treatment, Res could improve the spatial abilities of the mice to a certain extent and also increase the expression of SOD, GSH-Px, CAT, and HO-1 at the mRNA level (P<0.05). In addition, enhanced SOD, GSH-Px, and CAT activities and HO-1 protein levels and decreased MDA content (P<0.05) were detected in the brain tissue of the Res-treated mice. The cytoplasmic Nrf2 content in the Res-treated mice was also decreased while the nuclear Nrf2 content and the nuclear translation rate of Nrf2 were increased (P<0.05). Res could decrease the expression of ERβ in the brain tissue at the mRNA and protein levels and the expression of Aβ in the brain tissue at the protein level. Res could also increase the mRNA and protein expression of ERα and ChAT and the protein expression of estradiol in the brain tissue. Conclusion Res can increase the antioxidant capacity of AD models through the Nrf2/HO-1 signaling pathway. In addition, Res can enhance estrogen levels in an AD model. These findings provide a new idea for the treatment of AD.

Arsenic-Induced Neurotoxicity by Dysfunctioning Cholinergic and Dopaminergic System in Brain of Developing Rats.

Chronic exposure to arsenic via drinking water throughout the globe is assumed to cause a developmental neurotoxicity. Here, we investigated the effect of perinatal arsenic exposure on the neurobehavioral and neurochemical changes in the corpus striatum, frontal cortex, and hippocampus that is critically involved in motor and cognition functions. In continuation of previous studies, this study demonstrates that perinatal exposures (GD6-PD21) to arsenic (2 or 4mg/kg body weight, p.o.) cause hypo-activity in arsenic-exposed rats on PD22. The hypo-activity was found to be linked with a decrease in the mRNA and protein expression of the DA-D2 receptor. Further, a protein expression of tyrosine hydroxylase (TH), levels of dopamine, and its metabolites were also significantly impaired in corpus striatum. The arsenic-exposed groups showed spatial learning and memory significantly below the average in a dose-dependent manner for the controls. Here, we evaluated the declined expression of CHRM2 receptor gene and protein expression of ChAT, PKCβ-1 in the frontal cortex and hippocampus, which are critically involved in cognition functions including learning and memory. A trend of recovery was found in the cholinergic and dopaminergic system of the brain, but changes remained persisted even after the withdrawal of arsenic exposure on PD45. Taken together, our results indicate that perinatal arsenic exposure appears to be critical and vulnerable as the development of cholinergic and dopaminergic system continues during this period.

Edaravone attenuates intracerebroventricular streptozotocin-induced cognitive impairment in rats.

Alzheimer's disease is a major cause of dementia worldwide. Edaravone, a potent free radical scavenger, is reported to be neuroprotective. The present study was designed to investigate the effect of chronic edaravone administration on intracerebroventricular-streptozotocin (ICV-STZ) induced cognitive impairment in male Wistar rats. Cognitive impairment was developed by single ICV-STZ (3mg/kg) injection bilaterally on day 1. Edaravone (1, 3 and 10mg/kg, orally, once daily) was administered for 28days. Morris water maze and passive avoidance tests were used to assess cognitive functions at baseline and on days 14 and 28. ICV-STZ caused cognitive impairment as evidenced by increased escape latency and decreased time spent in target quadrant in the Morris water maze test and reduced retention latency in the passive avoidance test. STZ caused increase in oxidative stress, cholinesterases, inflammatory cytokines and protein expression of ROCK-II and decrease in protein expression of ChAT. Edaravone ameliorated the STZ-induced cognitive impairment. STZ-induced increase in oxidative stress and increased levels of pro-inflammatory cytokines (TNF-α, IL-1β) were mitigated by edaravone. Edaravone also prevented STZ-induced increased protein expression of ROCK-II. Moreover, edaravone significantly prevented STZ-induced increased activity of cholinesterases in the cortex and hippocampus. The decreased expression of ChAT caused by STZ was brought towards normal by edaravone in the hippocampus. The results thus show that edaravone is protective against STZ-induced cognitive impairment, oxidative stress, cholinergic dysfunction and altered protein expressions. This study thus suggests the potential of edaravone as an adjuvant in the treatment of Alzheimer's disease.

Brain cholinergic alterations in rats subjected to repeated immobilization or forced swim stress on lambda-cyhalothrin exposure

Role of immobilization stress (IMS), a psychological stressor and forced swim stress (FSS), a physical stressor was investigated on the neurobehavioral toxicity of lambda-cyhalothrin (LCT), a new generation type-II synthetic pyrethroid. Pre-exposure of rats to IMS (15 min/day) or FSS (3 min/day) for 28 days on LCT (3.0 mg/kg body weight, p.o.) treatment for 3 days resulted to decrease spatial learning and memory and muscle strength associated with cholinergic-muscarinic receptors in frontal cortex and hippocampus as compared to those exposed to IMS or FSS or LCT alone. Decrease in acetylcholinesterase activity, protein expression of ChAT and PKC-β1 associated with decreased mRNA expression of CHRM2, AChE and ChAT in frontal cortex and hippocampus was also evident in rats pre-exposed to IMS or FSS on LCT treatment, compared to rats exposed to IMS or FSS or LCT alone. Interestingly, changes both in behavioral and neurochemical endpoints were marginal in rats subjected to IMS or FSS for 28 days or those exposed to LCT for 3 days alone, compared to controls. The results suggest that stress is an important contributor in LCT induced cholinergic deficits.

REST/NRSF-induced changes of ChAT protein expression in the neocortex and hippocampus of the 3xTg-AD mouse model for Alzheimer 's disease

AimsThe cholinergic system is one of the neurotransmitter systems altered in Alzheimer's disease (AD), the most common form of human dementia. The objective of this work was to determine the REST/NRSF involvement in altered ChAT expression in the neocortex and hippocampus of an AD transgenic mouse (homozygous 3xTg-AD) that over-expresses 3 proteins, amyloid-β precursor protein, presenilin-1, and tau, all of which are associated with AD and cause cellular degeneration. Main methodsTwo groups (WT and 3xTg-AD) of 11-month-old female mice were analyzed and compared. Half of the brains of each group were used for ChAT immunohistochemistry, and Western Blot analyses of ChAT and REST/NRSF were performed on the other half. Key findingsWe observed significant decreases in the number of ChAT-immunoreactive cells in the Meynert nucleus and of fibers in the frontal motor cortex and hippocampal CA1 area in transgenic mice compared with control mice. An increased level of REST/NRSF protein and a reduction of ChAT protein expression in the 3xTg-AD mice compared with their controls were also found in both in the latter two cerebral regions. SignificanceThe increased REST/NRSF expression reported here and its effect on the regulatory region for ChAT transcription could explain the decreased expression of ChAT in the 3xTg-AD mouse; these findings may be associated with the degeneration observed in AD.

Neural restrictive silencer factor and choline acetyltransferase expression in cerebral tissue of Alzheimer's Disease patients: a pilot study

Decreased Choline Acetyltransferase (ChAT) brain level is one of the main biochemical disorders in Alzheimer’s Disease (AD). In rodents, recent data show that the CHAT gene can be regulated by a neural restrictive silencer factor (NRSF). The aim of the present work was to evaluate the gene and protein expression of CHAT and NRSF in frontal, temporal, entorhinal and parietal cortices of AD patient brains. Four brains from patients with AD and four brains from subjects without dementia were studied. Cerebral tissues were obtained and processed by the guanidine isothiocyanate method for RNA extraction. CHAT and NRSF gene and protein expression were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. CHAT gene expression levels were 39% lower in AD patients as compared to the control group (p < 0.05, U test). ChAT protein levels were reduced by 17% (p = 0.02, U test). NRSF gene expression levels were 86% higher in the AD group (p = 0.001, U test) as compared to the control group. In the AD subjects, the NRSF protein levels were 57% higher (p > 0.05, U test) than in the control subjects. These findings suggest for the first time that in the brain of AD patients high NRSF protein levels are related to low CHAT gene expression levels.

A Non-Neuronal Cardiac Cholinergic System Plays a Protective Role in Myocardium Salvage during Ischemic Insults

BackgroundIn our previous study, we established the novel concept of a non-neuronal cardiac cholinergic system–cardiomyocytes produce ACh in an autocrine and/or paracrine manner. Subsequently, we determined the biological significance of this system–it played a critical role in modulating mitochondrial oxygen consumption. However, its detailed mechanisms and clinical implications have not been fully investigated.AimWe investigated if this non-neuronal cardiac cholinergic system was upregulated by a modality other than drugs and if the activation of the system contributes to favorable outcomes.ResultsCholine acetyltransferase knockout (ChAT KO) cells with the lowest cellular ACh levels consumed more oxygen and had increased MTT activity and lower cellular ATP levels compared with the control cells. Cardiac ChAT KO cells with diminished connexin 43 expression formed poor cell–cell communication, evidenced by the blunted dye transfer. Similarly, the ChAT inhibitor hemicholinium-3 decreased ATP levels and increased MTT activity in cardiomyocytes. In the presence of a hypoxia mimetic, ChAT KO viability was reduced. Norepinephrine dose-dependently caused cardiac ChAT KO cell death associated with increased ROS production. In in vivo studies, protein expression of ChAT and the choline transporter CHT1 in the hindlimb were enhanced after ischemia-reperfusion compared with the contralateral non-treated limb. This local effect also remotely influenced the heart to upregulate ChAT and CHT1 expression as well as ACh and ATP levels in the heart compared with the baseline levels, and more intact cardiomyocytes were spared by this remote effect as evidenced by reduced infarction size. In contrast, the upregulated parameters were abrogated by hemicholinium-3.ConclusionThe non-neuronal cholinergic system plays a protective role in both myocardial cells and the entire heart by conserving ATP levels and inhibiting oxygen consumption. Activation of this non-neuronal cardiac cholinergic system by a physiotherapeutic modality may underlie cardioprotection through the remote effect of hindlimb ischemia-reperfusion.

Does senile impairment of cholinergic system in rats concern only disturbances in cholinergic phenotype or the progressive degeneration of neuronal cell bodies?

The trophic effect of continuous intraventricular infusion of nerve growth factor (NGF) on morphology of the basal forebrain (BF) cholinergic neurons was tested in 4- and 28-month-old male Wistar rats. All studies were conducted using behaviorally uncharacterized animals from the same breeding colony. Immunohistochemical procedure for choline acetyltransferase (ChAT) and p75NTR receptor has been applied to identify cholinergic cells in the structures of basal forebrain (BF). Using a quantitative image analyzer, morphometric and densitometric parameters of ChAT- and p75NTR-positive cells were measured immediately after cessation of NGF infusion. In 28-month-old non-treated rats the number of intensively ChAT-positive cells in all forebrain structures was reduced by 50-70% as compared with young animals. The remaining ChAT-positive cells appeared shrunken and the neuropil staining was NTR markedly reduced. In contrast, the same neurons when stained for p75 were numerous and distinctly visible with perfect morphology. Analysis of Nissl stained sections also showed that 28-month-old rats did not display significant losses of neuronal cell bodies. NGF restored the number of intensely stained ChAT-positive cells to about 90% of that for young controls and caused a significant increase in size of those cells in 28-month-old rats as compared with the control, age-matched group. NGF did not influence the morphology of p75NTR-positive neurons, which were well labeled, irrespective of treatment and age of the rats. In 4-month-old rats, NGF infusion decreased the intensity of both ChAT and p75NTR immunostaining. These data provide some evidence for preservation of BF cholinergic neurons from atrophy during aging and indicate that senile impairment of the cholinergic system in rats concerns decrease in ChAT-protein expression rather than an acute degeneration of neuronal cell bodies. Treatment with NGF resulted in restoration of cholinergic phenotype in the BF neurons of aged rats. However, the present study also rises issue of possible detrimental effects of NGF in young normal animals.