Alpha1 Subunit Research Articles

XPD mutations in trichothiodystrophy hamper collagen VI expression and reveal a role of TFIIH in transcription derepression

Mutations in the XPD subunit of the transcription/DNA repair factor (TFIIH) give rise to trichothiodystrophy (TTD), a rare hereditary multisystem disorder with skin abnormalities. Here, we show that TTD primary dermal fibroblasts contain low amounts of collagen type VI alpha1 subunit (COL6A1), a fundamental component of soft connective tissues. We demonstrate that COL6A1 expression is downregulated by the sterol regulatory element-binding protein-1 (SREBP-1) whose removal from the promoter is a key step in COL6A1 transcription upregulation in response to cell confluence. We provide evidence for TFIIH being involved in transcription derepression, thus highlighting a new function of TFIIH in gene expression regulation. The lack of COL6A1 upregulation in TTD is caused by the inability of the mutated TFIIH complexes to remove SREBP-1 from COL6A1 promoter and to sustain the subsequent high rate of COL6A1 transcription. This defect might account for the pathologic features that TTD shares with hereditary disorders because of mutations in COL6A genes.

Expression of Acetylcholine and Its Receptor in Human Sympathetic Ganglia in Primary Hyperhidrosis

The pathophysiologic characteristics of primary hyperhidrosis are not well understood and seem to be related to a sympathetic nervous system dysfunction. The resection of thoracic sympathetic chain ganglia is the most effective treatment for hyperhidrosis; however sympathetic ganglia function in normal individuals and in patients with hyperhidrosis is unknown. A cross-sectional study, in which 2 groups of 20 subjects were analyzed: the hyperhidrosis group (HYP), comprised of patients with hyperhidrosis who were eligible for thoracic sympathectomy, and the control group (CON) comprised of brain-dead organ donors without a history of hyperhidrosis. For each subject, the following were performed: resection of the third left sympathetic ganglion, measurement of the ganglion's diameter, and immunohistochemical evaluation by quantification of strong and weak expression areas of primary antibodies against acetylcholine and alpha-7 neuronal nicotinic receptor subunit. The presence of a strong alpha-7 subunit expression area was 4.85% in patients with primary hyperhidrosis and 2.34% in controls (p < 0.001), whereas the presence of a weak expression area was 11.48% in the HYP group and 4.59% in the CON group (p < 0.001). Strong acetylcholine expression was found in 4.95% of the total area in the HYP group and in 1.19% in the CON group (p < 0.001), whereas weak expression was found in 18.55% and 6.77% of the HYP and CON groups, respectively (p < 0.001). Furthermore, diameter of the ganglia was 0.71 cm in the HYP group and 0.53 cm in the CON group (p < 0.001). There is a higher expression of acetylcholine and alpha-7 neuronal nicotinic receptor subunit in the sympathetic ganglia of patients with hyperhidrosis. Furthermore, the diameter of the thoracic sympathetic chain ganglia is larger in such patients.

Expression of Integrin Alpha-3 and Beta-4 Subunits on the Process of Peri-Implant Epithelium Formation

Integrin, a component of the hemidesmosome, plays a role for epithelial cell migration and adhesion. This study investigated the process of peri-implant epithelium (PIE) formation after implantation, and compared it to the process of oral mucosa healing after tooth extraction. At the healing site of extraction socket without implant, the original junctional epithelium (JE) had disappeared at week 2, and the oral epithelium (OE) with integrin-α3 positive basal cells extending from the sides of the wound, then joined in the middle of the extraction socket. On the other hand in implant group, newly formed epithelium with integrin-α3 positive cells from the OE extended apically 1 week after implantation. After 3 weeks, basal cells of the new epithelium consisted of those with integrin-α3 positive but β4 negative. Finally, after 4 weeks, integrin-β4 was expressed at the implant-PIE interface. These findings suggest that integrin α3β1 plays a role in cell migration during PIE formation from OE. Furthermore, after the completion of PIE constitution, integrin α6β4 contributes to the attachment to titanium.

Neuronal cell-type specific DNA methylation patterns of the Cacna1c gene

Gene expression of the alpha-1 subunit of the L-type voltage-gated calcium channel, CACNA1C, is known to be complexly regulated. Because CACNA1C is not only a crucial gene in normal brain function but also a promising candidate risk gene for psychiatric disorders such as bipolar disorder and schizophrenia, elucidating the molecular basis of transcriptional regulatory mechanism will be critically important. Here we examined DNA methylation status of CpG islands and a CpG island shore on mouse Cacna1c in neuronal and non-neuronal nuclei, which were separated with a fluorescent activated cell sorting technique. We found that neurons and non-neurons showed differential DNA methylation profile on a CpG island shore. This difference was evolutionarily conserved in human neuronal and non-neuronal nuclei in the prefrontal cortex, suggesting that DNA methylation status on the CpG island shore of Cacna1c may have an important role in transcript regulation.

The GABAAreceptor α2 subunit gene (GABRA2) is associated with alcohol-related behavior

Background Gamma-aminobutyric acid-A (GABA-A) receptors, the major inhibitory neurotransmitter receptors in the brain are implicated in the acute and chronic effects of alcohol, including tolerance, dependence and withdrawal. Various polymorphisms in the gene encoding the GABA-A receptor alpha2 subunit (GABRA2) have been associated with alcoholism and with antisocial behavior in different populations of European ancestry. As early onset of alcoholism often reflects greater severity, including a higher risk for recurrence, comorbid antisocial personality disorder and conduct disorder, Cloninger’s classification distinguishes type II alcoholism with an early onset, elevated levels of antisocial behavior and delinquency, from the type I alcoholism with a late onset, neurotic symptoms and minimal criminality. Materials and methods Genotyping of GABRA2 polymorphisms (rs567926, rs279858 and rs9291283) was performed in samples of 355 alcoholic patients of Croatian origin (280 males and 75 females) using TaqMan Real-Time allelic discrimination technique after extraction of DNA from the whole blood. The results of allelic and haplotypic analysis were compared between alcohol-dependent subjects with a combination of early onset of alcohol abuse and presence of aggressive behavior corresponding to type II alcoholism subgroup, and individuals with the late onset of alcohol abuse and without aggression corresponding to type I alcoholism subgroup, according to Cloninger. Results Cloninger’s Type I and Type II alcohol-dependent patients did not differ significantly in the frequency of the genotypes and alleles for rs567926, rs279858 or rs9291283. However, the G-T- G haplotype was more often present in the alcohol- dependent subjects with early onset of alcohol abuse and aggressive behavior, corresponding to the Cloninger type II alcoholism subgroup (χ2 = 6.102, p = 0.013). Conclusions Our results revealed a haplotypic association between the GABRA2 gene and a more severe form of alcoholism, characterized by the early onset of alcohol abuse and presence of aggressive behavior. These findings support an important role of GABA-A receptors in the susceptibility to alcoholism and highlight them as potential targets for novel therapeutics in the treatment of alcohol dependence.

Radixin regulates gamma-aminobutyric acid-A receptors in human granulosa cells

Gamma-aminobutyric acid (GABA) acts as an inhibitory transmitter in mature central nervous system. GABA/GABA-A receptors are also present in non-neural tissue, including ovary. We have shown a receptor specific effect of GABA-A on aromatase (CYP19) induction in cultured human granulosa cells (GC), which requires the presence of insulin. The 81-kDa actin-binding protein radixin (RDX) from ezrin/radixin/moesin family has been shown to directly link the alpha-5 subunit of GABA-A receptor to the cytoskeleton acts as membrane organizer and linker.

Genetic association of GABA-A receptor alpha-2 and mu opioid receptor with cocaine cue-reactivity: evidence for inhibitory synaptic neurotransmission involvement in cocaine dependence.

This pilot feasibility study examined the role of genetics in laboratory-induced cocaine craving. Thirty-four African American, cocaine-depend- ent male subjects underwent a baseline assessment, cue-exposure session, and genetic analysis. Subjects were classified as either cue-reactive or nonreactive. Among single nucleotide polymorphism markers in 13 candidate genes examined for association with cocaine cue-reactivity, two were statistically significant: GABRA2 (coding for GABA-A receptor alpha-2 subunit; rs11503014, nominal p= .001) and OPRM1 (coding for mu opioid receptor; rs2236256, nominal p= .03). These pilot results suggest that cocaine craving shows variability among cocaine-dependent subjects, and that GABRA2 and OPRM1 polymorphisms have differential influences on cocaine cue-reactivity, warranting studies in future research.

Corticosterone attenuates conditioned fear responses and potentiates the expression of GABA-A receptor alpha-2 subunits in the brain structures of rats selected for high anxiety

The aim of the experiment was to assess the effects of an acutely administered corticosterone on the expression of GABA-A receptor alpha-2 subunits in the brain structures of high (HR) and low (LR) anxiety rats (divided according to their conditioned fear-induced freezing response) subjected to a second conditioned fear session (1week after fear conditioning). We found that corticosterone (20mg/kg, sc) given to rats prior to the second conditioned fear session significantly enhanced a decrease in fear expression in the HR group. The behavioural effect of fear was accompanied by the increased expression of alpha-2 subunits in the basolateral amygdala (BLA) and the dentate gyrus of the hippocampus (DG) of the HR group. Corticosterone potentiated the effect of fear on alpha-2 subunit expression in the BLA, DG, the cingulate cortex area 1 and the secondary motor cortex (areas Cg1 and M2). The current study provides insight into the mechanisms that may be responsible for the beneficial effects of glucocorticoids in the therapy of some anxiety disorders.

Differential expression of proteins in Leishmania (Viannia) panamensis associated with mechanisms of resistance to meglumine antimoniate

Alzheimer's disease is a multifactorial disease affecting approximately twenty million people worldwide. Numerous variables are associated with increased risk of developing this severe neurological disorder. Among the risk factors, diabetes mellitus, and the ε4 isoform of the APOE gene have been amply demonstrated as increasing the risk of developing this disease. To determine if a correlation exists between APOE genotype, diabetes mellitus and Alzheimer's disease. Clinical studies were carried out by surveying the clinical histories in a group of patients in the province of Antioquia, Colombia. Forty-three Alzheimer's patients were compared with 43 control subjects, paired by age and gender. Commercially available methods were used to determine whether the patients had diabetes, and restriction enzyme-based genotyping was used to determine the APOE genotypes. The most common non-neurological comorbidities were: arterial hypertension, acute myocardial infarction, chronic obstructive pulmonary disease and hypothyroidism. From the many variables investigated, two were conclusive: (1) the presence of Alzheimer's disease was higher in patients with diabetes mellitus, and (2) no correlation between late-onset sporadic Alzheimer's disease and APOE was found in the target population. To detect any association with the APOE genotype, a study involving much a larger population samples must be undertaken.

CDNA Cloning and Expression Analysis of the Nicotinic Acetylcholine Receptor Alpha6 Subunit in the Oriental Fruit Fly,Bactrocera dorsalis(Diptera: Tephritidae)

Nicotinic acetylcholine receptors (nAChR) play major roles in the fast synapse cholinergic transmission in the insect central nervous system, and they are recognized as potential target sites for insecticides. In this study, the full-length cDNA sequence of the nAChR alpha6 subunit (Bdorα6) was isolated and characterized from the oriental fruit fly, Bactrocera dorsalis, using the Rapid Amplification of cDNA Ends (RACE) technique. The Bdorα6 cDNA was 2020 bp long, containing an open reading frame of 1467 bp, which encodes 488 amino acid residues with a predicated molecular mass of 55.6 kDa. Homology analysis indicated that Bdorα6 has typical features of a nAChR α subunit. Phylogenetic analysis with other insect nAChR subunits suggested that Bdorα6 was most closely related to Dα6 of Drosophila melanogaster. The developmental changes and tissue-specificity of the relative expression levels of Bdorα6 were investigated using quantitative real-time PCR. The highest expression level was observed in the first instar larva and the lowest was in the pupa, and there was a descending expression level during the developmental period from the first instar larva to the pupa. The relative expression levels were 23-, 39-, 6-, 2- and 27-fold higher in the egg, first, second and third instar larvae and adult than in the pupa, respectively. Furthermore, Bdorα6 was expressed in the head and thorax at 109- and 15-fold higher levels than in the abdomen. The results provided some important baseline data for further molecular studies in Bactrocera dorsalis.

Psychiatric and Neurocognitive Morbidity in Rapid-Onset Dystonia Parkinsonism (P01.225)

Objective: Elevated rates of psychiatric disease and cognitive dysfunction have been found in primary dystonias. These features have not been studied in Rapid-Onset Dystonia-Parkinsonism (RDP), DYT12. Data are presented for 47 individuals (with or without gene mutation). Background RDP is a rare form of dystonia caused by mutation of the gene encoding the alpha-3 subunit of Na+/K+-ATPase ( ATP1A3 ). RDP is characterized by abrupt onset of dystonic symptoms with features of Parkinsonism and subsequent stabilization without recovery. Psychiatric and cognitive symptoms have not been systematically studied in RDP. Design/Methods: The SCID-I and CIDI-I were administered to 47 individuals, including 27 mutation carriers (24 motor-manifesting, 3 non-motor-manifesting) and 20 family members without ATP1A3 mutation. Discrepancies in diagnoses were adjudicated by a psychiatrist blinded to mutation status, who rendered final diagnoses. Cognitive data were obtained on 43 individuals, including 23 motor-manifesting carriers, 3 non-motor-manifesting carriers, and 17 non-carrier relatives. Results: Mood disorders appear more prevalent among carriers (44%) compared to non-carriers (25%). Anxiety (44% vs. 50%) and substance use (33% vs. 32%) were similar across groups. Five individuals from three families with ATP1A3 mutation (19%) exhibited psychotic disorder. Psychotic disorder was not present among non-carriers. One non-carrier exhibited evidence of visual memory dysfunction. Gene carriers exhibited visual (17%) and verbal (22%) memory deficits. More gene carriers exhibited impairment compared to non-carriers on Trail Making B (54% vs. 12%) and Trail Making A (42% vs. 29%). Self reported rates of childhood learning difficulties (post-RDP onset) were higher among gene carriers (69% vs. 24%). Conclusions: Anxiety disorders and substance use/abuse did not differ among individuals with and without ATP1A3 mutation. Increased mood and psychotic disorders for gene carriers illustrate comorbid mental illness. Impaired memory and executive functioning is consistent with findings in other dystonias. Higher prevalence of childhood learning difficulties in gene carriers suggests non-motor manifestations prior to motor onset. Supported by: NINDS 5R01NS058949-03. Disclosure: Dr. Brashear has received personal compensation for activities with Allergan, Inc., Merz Pharma, Ipsen as a consultant. Dr. Brashear has received personal compensation in an editorial capacity for the American Academy of Neurology. Dr. Brashear will receive future royalty payments for editing a book on spasticity. Dr. Brasher has received research support from Allergan, Inc., Merz Pharma, and Ipsen. Dr. Cook has nothing to disclose. Dr. Amponsah has nothing to disclose. Dr. Hill has nothing to disclose. Dr. Snively has nothing to disclose. Dr. Light has nothing to disclose. Dr. Suerken has nothing to disclose. Dr. McCall has received personal compensation for activities with Sepracor, Inc. Dr. Boggs has nothing to disclose. Dr. Stacy has received personal compensation for activities with Allergan, Inc., Biogen Idec, General Electric, Novartis, Osmotica, Teva Neuroscience, and Synosia. Dr. Stacy has received research support from Ceregene, Impax, Neuraltus, Novartis, Schering-Plough, and the Parkinson Study Group. Dr. Ozelius has nothing to disclose. Dr. Sweadner has nothing to disclose.

Antibody to α4 integrin suppresses natural killer cells infiltration in central nervous system in experimental autoimmune encephalomyelitis

Natalizumab inhibits the influx of leukocytes into the central nervous system (CNS) via blockade of alpha-4 subunit of very late activation antigen (VLA)-4. The association of natalizumab therapy with progressive multifocal leukoencephalopathy (PML) suggests a disturbance of CNS immune surveillance in a small percentage of Multiple Sclerosis (MS) patients exposed to the medication. Natural killer (NK) cells are known to play an important role in modulating the evolution of different phases of this lymphocyte mediated disease, and we investigated the effects of natalizumab on the NK cell phenotype and infiltration in the CNS in experimental autoimmune encephalomyelitis (EAE), a murine model of MS. Our data show that both resting (from naïve mice) and activated (from EAE mice) NK cells express high levels of VLA-4, and anti-VLA-4 antibody treatment significantly decreases NK cells frequency in the CNS of EAE mice. Moreover, we find that anti-VLA-4 possibly impairs NK cells migratory potential, since unblocked VLA-4 expression levels were downregulated on those NK cells that penetrate the CNS. These data suggest that treatment with antibody to VLA-4 may alter immune surveillance of the CNS by impacting NK cell functions and might contribute to the understanding of the mechanisms leading to the development of PML in some MS patients.

The association of the alpha-5 subunit of the nicotinic acetylcholine receptor gene and the brain-derived neurotrophic factor gene with different aspects of smoking behavior

Recent studies show that different aspects of smoking behavior are associated with the α-5 subunit of the nicotinic acetylcholine receptor (CHRNA5) gene and the gene coding for brain-derived neurotrophic factor (BDNF). This raises the question whether the amount of cigarettes smoked per day has a different genetic background than smoking initiation and what other smoking phenotypes may be relevant. The aim of this study was to replicate these associations in a large population-based sample. We investigated the association with smoking initiation and the number of cigarettes used per day and additional smoking phenotypes in a population-based sample of 2166 participants of Dutch origin. Rs6265 in BDNF was not associated with smoking initiation. This single nucleotide polymorphism was associated with smoking cessation. Rs16969968 in CHRNA5 was associated with the amount of nicotine used and in particular smoking 25 cigarettes or more per day. Overall, the results confirm the involvement of the CHRNA5 gene in the amount of nicotine use and further suggest involvement of the BDNF gene in smoking behavior.

Restoring Epilepsy‐Associated Mutant GABAA Receptor Proteostasis

GABAA (γ‐aminobutyric acid type A) receptors are major inhibitory neurotransmitter ligand‐gated ion channels in the mammalian central nervous system. Recently, several mutations in the GABAA receptors were identified to compromise protein folding, subjecting channel proteins to endoplasmic reticulum (ER)‐associated degradation, leading to epilepsy due to loss of channel function. One prominent example is the A322D mutation in the alpha1 subunit of GABAA receptor, resulting in juvenile myoclonic epilepsy. We employed a mass‐spectrometry based proteomics approach to quantify the proteome difference between wild type and alpha1(A322D)beta2gamma2 GABAA receptors. Our preliminary result showed that critical ER chaperones, namely BiP and calnexin, interact differently with alpha1 subunit of GABAA receptors. Overexpression of BiP and calnexin increased the expression level of mutant GABAA receptors due to enhanced ER folding capacity. We propose that restoring GABAA receptor proteostasis may provide a novel therapeutic strategy for the amelioration of epilepsy and perhaps other misfolding‐associated channelopathies.

Association of single nucleotide polymorphisms of nicotinic acetylcholine receptor subunits with cervical neoplasia

AimsCholinergic signaling, particularly in response to non-physiological ligands like nicotine, stimulates carcinogenesis of a variety of tissue types including epithelia of the cervix uteri. Cholinergic signaling is mediated by nicotinic acetylcholine receptors (nAChRs), which are pentamers formed by subsets of 16 nAChR subunits. Recent literature suggests that single nucleotide polymorphisms (SNPs) of some of these subunits, notably alpha5, are risk factors for developing lung cancer in smokers as well as in non-smokers. Main methodsWe have studied the prevalence of four SNPs in the alpha5, alpha9, and beta1 subunits, which are expressed in cervical cells, in 456 patients with cervical cancers, precursor lesions, and healthy controls from two cohorts in Mexico. Key findingsA SNP in the alpha9 subunit, the G allele of rs10009228 (alpha9, A>G) shows a significant trend in the combined cohort, indicating that this allele constitutes a risk factor for neoplastic progression. The A allele of the SNP rs16969968 (alpha5, G>A), which correlates with the development of lung cancer, shows a non-significant trend to be associated with cervical lesions. Two other SNPs, rs55633891 (alpha9, C>T) and rs17856697 (beta1, A>G), did not exhibit a significant trend. SignificanceOur study points to a potential risk factor of cervical carcinogenesis with importance for DNA diagnosis and as a target for intervention.

Cardiotonic Steroids-Mediated Targeting of the Na+/K+-ATPase to Combat Chemoresistant Cancers

A large proportion of cancer patients fail to respond to conventional chemotherapy because of the intrinsic resistance of their cancer to pro-apoptotic stimuli and/or the acquisition of a multidrug resistant (MDR) phenotype during chronic chemotherapy. A new angle in chemotherapeutics against these cancer types associated with dismal prognoses would be the targeting of specific ion channels and pumps over expressed by cancer cells as compared to normal cells. Several reports suggest that the alpha subunits of the Na(+)/K(+)-ATPase (referred as sodium pump from now on) could be such targets, using cardiotonic steroids (CS) including cardenolides and bufadienolides. A significant proportion of non-small-cell-lung cancers (NSCLCs), glioblastomas (GBMs), melanomas and kidney cancers overexpresses the alpha-1 subunit of the sodium pump as compared to corresponding normal tissues, while colon cancers overexpress the alpha-3 subunit. Thus, a deeper knowledge of the structure-activity relationship (SAR), in terms of CS-mediated anticancer effects, to the sodium pump alpha subunits might enable the identification of potent anticancer agents with limited cardiotoxicity. The current review provides an in depth SAR analysis with respect to cardenolide- versus bufadienolide-mediated anticancer effects. Moreover, pharmacological data from in vitro and in vivo experiments, as well as pre-clinical and clinical trials regarding cardenolides to combat cancers associated with dismal prognoses are presented.

Function of nAChRs Containing Alpha5 Subunits

Neuronal nicotinic acetylcholine receptors (nAChRs) are abundantly expressed in the central nervous system. Neurons in several brain regions co-express alpha5, alpha4 and beta2 subunits. Changes in the receptor composition and stoichiometry of alpha4 and beta2 containing nAChRs are pertinent to disease states. Several genome-wide association and candidate gene studies have identified polymorphisms in the gene for the alpha5 subunit that are linked to an increased risk for nicotine addiction, alcohol addiction and lung cancer. However more information is required about the functional effects of incorporating a normal or mutated alpha5 subunit into the alpha4beta2 receptor pentamer. Here we examine differences in I-V relationships and dose-response relations between alpha4beta2 receptors and alpha4beta2 receptors including alpha5 subunits containing various mutations to the M2 domain expressed in Xenopus oocytes.

Alpha6∗ Nicotinic Receptors and P2X Receptors: Physical and Functional Interactions

Several neuronal cell types co-express nicotinic acetylcholine receptors (nAChRs) and P2X receptors (P2XRs); and previous experiments from several laboratories show that these two ligand-gated channel subtypes interact with each other, both functionally and physically. We extended these studies to interactions between alpha6 subunit-containing (alpha6∗) nAChRs and P2X2 or P2X3 receptors (P2X2Rs, P2X3Rs). In Xenopus oocytes, alpha6 subunits mutated in the M2 region co-expressed with beta4 subunits displayed ACh-induced currents. P2X2 or P2X3 receptors expressed in the same oocytes displayed ATP-induced currents. When ACh and ATP were applied simultaneously, the total current was less than the sum of the individual currents_the conventional definition of cross-inhibition_for alpha6beta4-P2X2 oocytes. Several combinations of fluorescent protein (FP)-fused alpha6, beta4, P2X2, and P2X3 subunits were expressed in cultured mouse cortical cells and Neuro2a cells. In Forster resonance energy transfer (FRET) measurements using fluorescent lifetime imaging microscopy (FLIM) and normalized FRET (NFRET), we detected physical contact between alpha6beta2 nAChRs and P2X2Rs, between alpha6beta4 nAChRs and P2X2Rs, and between alpha6beta4 nAChRs and P2X3Rs. Thus, two lines of evidence show interactions between alpha6∗ nAChRs and P2XRs.Support: Beckman fellowship to CIR, NIH (NRSA to CIR, DA-19375, NS034407), Wellcome Trust, Yousef Jameel Award.

A New View on Allosteric Modulation of Alpha 7 Nicotinic Acetylcholin Receptors

Nicotinic acetylcholine receptors (nAChR) are ligand gated cation channels that are opened by agonists like Acetylcholine and Nicotine. They are widely distributed in neurons all over central nervous system, where they are mainly found at postsynaptic locations of cholinergic nerve endings.The most abundant homomeric nicotinic acetylcholine receptors in the mammalian brain are the pentameric alpha7 nAChRs which consist of five alpha7 subunits. Each subunit provides an orthosteric low affinity binding site for its endogenous ligand, acetylcholine. Several lines of evidence suggest a link between the alpha7 neuronal nicotinic acetylcholine receptor and brain disorders including schizophrenia, Alzheimer's disease, and traumatic brain injury. Alpha 7 receptors are also a target for non-CNS indications like diabetes and asthma.View Large Image | View Hi-Res Image | Download PowerPoint SlideHere we show the results of the screen of allosteric modulators on alpha 7 receptors. High parallelization without a compromise in data quality combined with fast and accurate solution exchange allows characterization of many compounds. A second analysis at physiological temperatures showed a pronounced effect on the allosteric modulation.

N-cadherin induces partial differentiation of cholinergic presynaptic terminals in heterologous cultures of brainstem neurons and CHO cells

N-cadherin is a calcium-sensitive cell adhesion molecule commonly expressed at synaptic junctions and contributes to formation and maturation of synaptic contacts. This study used heterologous cell cultures of brainstem cholinergic neurons and transfected Chinese Hamster Ovary (CHO) cells to examine whether N-cadherin is sufficient to induce differentiation of cholinergic presynaptic terminals. Brainstem nuclei isolated from transgenic mice expressing enhanced green fluorescent protein (EGFP) under the control of choline acetyltransferase (ChAT) transcriptional regulatory elements (ChATBACEGFP) were cultured as tissue explants for 5 days and cocultured with transfected CHO cells for an additional 2 days. Immunostaining for synaptic vesicle proteins SV2 and synapsin I revealed a ~3-fold increase in the area of SV2 immunolabeling over N-cadherin expressing CHO cells, and this effect was enhanced by coexpression of p120-catenin. Synapsin I immunolabeling per axon length was also increased on N-cadherin expressing CHO cells but required coexpression of p120-catenin. To determine whether N-cadherin induces formation of neurotransmitter release sites, whole-cell voltage-clamp recordings of CHO cells expressing α3 and β4 nicotinic acetylcholine receptor (nAChR) subunits in contact with cholinergic axons were used to monitor excitatory postsynaptic potentials (EPSPs) and miniature EPSPs (mEPSPs). EPSPs and mEPSPs were not detected in both, control and in N-cadherin expressing CHO cells in the absence or presence of tetrodotoxin (TTX). These results indicate that expression of N-cadherin in non-neuronal cells is sufficient to initiate differentiation of presynaptic cholinergic terminals by inducing accumulation of synaptic vesicles; however, development of readily detectable mature cholinergic release sites and/or clustering of postsynaptic nAChR may require expression of additional synaptogenic proteins.