Α1 Subunits Research Articles

A Review of Genetic and Physiological Disease Mechanisms Associated With Cav1 Channels: Implications for Incomplete Congenital Stationary Night Blindness Treatment.

Calcium channels are crucial to a number of cellular functions. The high voltage-gated calcium channel family comprise four heteromeric channels (Cav1.1-1.4) that function in a similar manner, but that have distinct expression profiles. Three of the pore-forming α1 subunits are located on autosomes and the forth on the X chromosome, which has consequences for the type of pathogenic mutation and the disease mechanism associated with each gene. Mutations in this family of channels are associated with malignant hyperthermia (Cav1.1), various QT syndromes (Cav1.2), deafness (Cav1.3), and incomplete congenital stationary night blindness (iCSNB; Cav1.4). In this study we performed a bioinformatic analysis on reported mutations in all four Cav α1 subunits and correlated these with variant frequency in the general population, phenotype and the effect on channel conductance to produce a comprehensive composite Cav1 mutation analysis. We describe regions of mutation clustering, identify conserved residues that are mutated in multiple family members and regions likely to cause a loss- or gain-of-function in Cav1.4. Our research highlights that therapeutic treatments for each of the Cav1 channels will have to consider channel-specific mechanisms, especially for the treatment of X-linked iCSNB.

Integrin α3β1 Promotes Invasive and Metastatic Properties of Breast Cancer Cells through Induction of the Brn-2 Transcription Factor.

Simple SummaryMetastatic triple-negative breast cancer (TNBC) is highly lethal with limited therapy options. Integrin α3β1 is a cell surface receptor that interacts with the extracellular matrix and facilitates communication between tumor cells and their microenvironment. α3β1 is implicated in breast cancer progression and metastasis, so understanding mechanisms by which α3β1 promotes invasion and metastasis will facilitate the development of this integrin as a potential therapeutic target. Here we identify a novel role for α3β1 in promoting the expression of the transcription factor Brain-2 (Brn-2) in triple-negative breast cancer cells. We further report that Brn-2 promotes invasion and metastasis and partially restores invasion to cells in which expression of α3β1 has been suppressed. Bioinformatic analysis of patient datasets revealed a positive correlation between the expression of the genes encoding the integrin α3 subunit and Brn-2. In summary, our work identifies α3β1-mediated induction of Brn-2 as a mechanism that regulates invasive and metastatic properties of breast cancer cells.In the current study, we demonstrate that integrin α3β1 promotes invasive and metastatic traits of triple-negative breast cancer (TNBC) cells through induction of the transcription factor, Brain-2 (Brn-2). We show that RNAi-mediated suppression of α3β1 in MDA-MB-231 cells caused reduced expression of Brn-2 mRNA and protein and reduced activity of the BRN2 gene promoter. In addition, RNAi-targeting of Brn-2 in MDA-MB-231 cells decreased invasion in vitro and lung colonization in vivo, and exogenous Brn-2 expression partially restored invasion to cells in which α3β1 was suppressed. α3β1 promoted phosphorylation of Akt in MDA-MB-231 cells, and treatment of these cells with a pharmacological Akt inhibitor (MK-2206) reduced both Brn-2 expression and cell invasion, indicating that α3β1-Akt signaling contributes to Brn-2 induction. Analysis of RNAseq data from patients with invasive breast carcinoma revealed that high BRN2 expression correlates with poor survival. Moreover, high BRN2 expression positively correlates with high ITGA3 expression in basal-like breast cancer, which is consistent with our experimental findings that α3β1 induces Brn-2 in TNBC cells. Together, our study demonstrates a pro-invasive/pro-metastatic role for Brn-2 in breast cancer cells and identifies a role for integrin α3β1 in regulating Brn-2 expression, thereby revealing a novel mechanism of integrin-dependent breast cancer cell invasion.

Possible correlated variation of GABAA receptor α3 expression with hippocampal cholinergic neurostimulating peptide precursor protein in the hippocampus

Cholinergic neural activation from the medial septal nucleus to hippocampus plays a crucial role in episodic memory as a regulating system for glutamatergic neural activation in the hippocampus. As a candidate regulating factor for acetylcholine synthesis in the medial septal nucleus, hippocampal cholinergic neurostimulating peptide (HCNP) was purified from the soluble fraction of young adult rat hippocampus. HCNP is released from its precursor protein (HCNP-pp), also referred to as phosphatidylethanolamine-binding protein 1. We recently reported that HCNP-pp conditional knockout (KO) mice, in which the HCNP-pp gene was knocked out at 3 months of age by tamoxifen injection, display no significant behavioral abnormalities, whereas HCNP-pp KO mice have a diminished cholinergic projection to CA1 and a decreased of theta activity in CA1. In this study, to address whether HCNP-pp reduction in early life is associated with behavioral changes, we evaluated the behavior of HCNP-pp KO mice in which HCNP-pp was downregulated from an early phase (postnatal days 14–28). As unexpected, HCNP-pp KO mice had no behavioral deficits. However, a significant positive correlation between HCNP-pp and gamma-aminobutyric acid A (GABAA) receptor α3 subunit mRNA expression was found in individuals. This finding suggests involvement of HCNP-pp in regulating GABAA receptor α3 gene expression.

C1GALT1 high expression is associated with poor survival of patients with pancreatic ductal adenocarcinoma and promotes cell invasiveness through integrin \u03b1v

Pancreatic adenocarcinoma (PDAC) is a leading cause of cancer-related death. Altered glycosylation contributes to tumor progression and chemoresistance in many cancers. C1GALT1 is the key enzyme controlling the elongation of GalNAc-type O-glycosylation. Here we showed that C1GALT1 was overexpressed in 85% (107/126) of PDAC tumors compared with adjacent non-tumor tissues. High expression of C1GALT1 was associated with poor disease-free and overall survival (n = 99). C1GALT1 knockdown using siRNA suppressed cell viability, migration, and invasion as well as increased gemcitabine sensitivity in PDAC cells. In contrast, C1GALT1 overexpression enhanced cell migration and invasion. In subcutaneous and pancreatic orthotopic injection models, C1GALT1 knockdown decreased tumor growth and metastasis of PDAC cells in NOD/SCID mice. Mechanistically, C1GALT1 knockdown dramatically suppressed cell-extracellular matrix (ECM) adhesion, which was associated with decreased phosphorylation of FAK at Y397/Y925 and changes in O-glycans on integrins including the β1, αv, and α5 subunits. Using functional blocking antibodies, we identified integrin αv as a critical factor in C1GALT1-mediated invasiveness of PDAC cells. In conclusion, this study not only reveals that C1GALT1 could be a potential therapeutic target for PDAC but also provides novel insights into the role of O-glycosylation in the α subunits of integrins.

Αlpha 5 subunit-containing GABAA receptors in temporal lobe epilepsy with normal MRI.

GABAA receptors containing the α5 subunit mediate tonic inhibition and are widely expressed in the limbic system. In animals, activation of α5-containing receptors impairs hippocampus-dependent memory. Temporal lobe epilepsy is associated with memory impairments related to neuron loss and other changes. The less selective PET ligand [11C]flumazenil has revealed reductions in GABAA receptors. The hypothesis that α5 subunit receptor alterations are present in temporal lobe epilepsy and could contribute to impaired memory is untested. We compared α5 subunit availability between individuals with temporal lobe epilepsy and normal structural MRI (‘MRI-negative’) and healthy controls, and interrogated the relationship between α5 subunit availability and episodic memory performance, in a cross-sectional study. Twenty-three healthy male controls (median ± interquartile age 49 ± 13 years) and 11 individuals with MRI-negative temporal lobe epilepsy (seven males; 40 ± 8) had a 90-min PET scan after bolus injection of [11C]Ro15-4513, with arterial blood sampling and metabolite correction. All those with epilepsy and six controls completed the Adult Memory and Information Processing Battery on the scanning day. ‘Bandpass’ exponential spectral analyses were used to calculate volumes of distribution separately for the fast component [VF; dominated by signal from α1 (α2, α3)-containing receptors] and the slow component (VS; dominated by signal from α5-containing receptors). We made voxel-by-voxel comparisons between: the epilepsy and control groups; each individual case versus the controls. We obtained parametric maps of VF and VS measures from a single bolus injection of [11C]Ro15-4513. The epilepsy group had higher VS in anterior medial and lateral aspects of the temporal lobes, the anterior cingulate gyri, the presumed area tempestas (piriform cortex) and the insulae, in addition to increases of ∼24% and ∼26% in the ipsilateral and contralateral hippocampal areas (P < 0.004). This was associated with reduced VF:VS ratios within the same areas (P < 0.009). Comparisons of VS for each individual with epilepsy versus controls did not consistently lateralize the epileptogenic lobe. Memory scores were significantly lower in the epilepsy group than in controls (mean ± standard deviation −0.4 ± 1.0 versus 0.7 ± 0.3; P = 0.02). In individuals with epilepsy, hippocampal VS did not correlate with memory performance on the Adult Memory and Information Processing Battery. They had reduced VF in the hippocampal area, which was significant ipsilaterally (P = 0.03), as expected from [11C]flumazenil studies. We found increased tonic inhibitory neurotransmission in our cohort of MRI-negative temporal lobe epilepsy who also had co-morbid memory impairments. Our findings are consistent with a subunit shift from α1/2/3 to α5 in MRI-negative temporal lobe epilepsy.

Association Between Susceptibility of Thrips palmi to Spinetoram and Frequency of G275E Mutation Provides Basis for Molecular Quantification of Field-Evolved Resistance.

Putative mechanisms underlying spinosyn resistance have been identified in controlled studies on many species; however, mechanisms underlying field-evolved resistance and the development of a molecular diagnostic method for monitoring field resistance have lagged behind. Here, we examined levels of resistance of melon thrips, Thrips palmi Karny (Thysanoptera:Thripidae), to spinetoram as well as target site mutations in field populations across China to identify potential mechanisms and useful molecular markers for diagnostic and quantifying purposes. In resistant populations, we identified the G275E mutation, which has previously been linked to spinosyns resistance, and F314V mutation, both located in the α6 subunit of the nicotinic acetylcholine receptor. There was a strong correlation between levels of spinetoram resistance and allele frequency of G275E mutation in field-collected populations (r2 = 0.84) and those reared under laboratory conditions for two to five generations (r2 = 0.91). LC50 ranged from 0.12 to 0.66 mg/liter in populations without G275E mutation, whereas it ranged from 33.12 to 39.91 mg/liter in most populations with a G275E mutation frequency more than 90%. Our results indicate that the field-evolved resistance of T. palmi to spinetoram in China is mainly conferred by the G275E mutation. The frequency of the G275E mutation provides a useful diagnostic for quantifying resistance levels in field populations of T. palmi.

Selective coactivation of α7- and α4β2-nicotinic acetylcholine receptors reverses beta-amyloid–induced synaptic dysfunction

Beta-amyloid (Aβ) has been recognized as an early trigger in the pathogenesis of Alzheimer's disease (AD) leading to synaptic and cognitive impairments. Aβ can alter neuronal signaling through interactions with nicotinic acetylcholine receptors (nAChRs), contributing to synaptic dysfunction in AD. The three major nAChR subtypes in the hippocampus are composed of α7-, α4β2-, and α3β4-nAChRs. Aβ selectively affects α7- and α4β2-nAChRs, but not α3β4-nAChRs in hippocampal neurons, resulting in neuronal hyperexcitation. However, how nAChR subtype selectivity for Aβ affects synaptic function in AD is not completely understood. Here, we showed that Aβ associated with α7- and α4β2-nAChRs but not α3β4-nAChRs. Computational modeling suggested that two amino acids in α7-nAChRs, arginine 208 and glutamate 211, were important for the interaction between Aβ and α7-containing nAChRs. These residues are conserved only in the α7 and α4 subunits. We therefore mutated these amino acids in α7-containing nAChRs to mimic the α3 subunit and found that mutant α7-containing receptors were unable to interact with Aβ. In addition, mutant α3-containing nAChRs mimicking the α7 subunit interact with Aβ. This provides direct molecular evidence for how Aβ selectively interacted with α7- and α4β2-nAChRs, but not α3β4-nAChRs. Selective coactivation of α7- and α4β2-nAChRs also sufficiently reversed Aβ-induced AMPA receptor dysfunction, including Aβ-induced reduction of AMPA receptor phosphorylation and surface expression in hippocampal neurons. Moreover, costimulation of α7- and α4β2-nAChRs reversed the Aβ-induced disruption of long-term potentiation. These findings support a novel mechanism for Aβ's impact on synaptic function in AD, namely, the differential regulation of nAChR subtypes.

The human-specific duplicated α7 gene inhibits the ancestral α7, negatively regulating nicotinic acetylcholine receptor-mediated transmitter release

Gene duplication generates new functions and traits, enabling evolution. Human-specific duplicated genes in particular are primary sources of innovation during our evolution although they have very few known functions. Here we examine the brain function of one of these genes (CHRFAM7A) and its product (dupα7 subunit). This gene results from a partial duplication of the ancestral CHRNA7 gene encoding the α7 subunit that forms the homopentameric α7 nicotinic acetylcholine receptor (α7-nAChR). The functions of α7-nAChR in the brain are well defined, including the modulation of synaptic transmission and plasticity underlying normal attention, cognition, learning, and memory processes. However, the role of the dupα7 subunit remains unexplored at the neuronal level. Here, we characterize that role by combining immunoblotting, quantitative RT-PCR and FRET techniques with functional assays of α7-nAChR activity using human neuroblastoma SH-SY5Y cell variants with different dupα7 expression levels. Our findings reveal a physical interaction between dupα7 and α7 subunits in fluorescent protein-tagged dupα7/α7 transfected cells that negatively affects normal α7-nAChR activity. Specifically, in both single cells and cell populations, the [Ca2+]i signal and the exocytotic response induced by selective stimulation of α7-nAChR were either significantly inhibited by stable dupα7 overexpression or augmented after silencing dupα7 gene expression with specific siRNAs. These findings identify a new role for the dupα7 subunit as a negative regulator of α7-nAChR-mediated control of exocytotic neurotransmitter release. If this effect is excessive, it would result in an impaired synaptic transmission that could underlie the neurocognitive and neuropsychiatric disorders associated with α7-nAChR dysfunction.

New perspectives of quercetin and vitamin C effects on fibronectin-binding integrins and chemokine receptors in prostate cancer cell lines.

The aim of this study is to investigate the effect of two abundant dietary supplements, quercetin and vitamin C on some factors involved in metastasis and proliferation of prostate cancer, which are resistant to conventional chemotherapies in late stages. Bone and brain are two common sites of metastases in prostate cancer, nevertheless the factors involved in their metastatic pathways are not well understood. The effect of quercetin (75µM) and vitamin C (100 µM) on CXCR4, CXCR7 chemokine receptors, α4, α5 and β1 integrins, ki-67 proliferation marker and Vascular endothelial growth factor, VEGF was evaluated using Quantitative Reverse Transcription PCR (RT-qPCR). The effect of quercetin and vitamin C alone was different on PC3 and DU145 prostate cancer cell lines, but sequential combination reduced significantly the expression of CXCR and CXCR7 chemokine receptors, α4, α5 and β1 integrin subunits, VEGF and Ki-67 proliferation markers in PC3 and DU145 cell lines. Our results indicated the beneficial effect of quercetin and vitamin C on prostate cancer cells with different metastatic sites and their differential response to the treatment which in turn may lead us to reach suitable therapeutic outcomes to combat cancer (Fig. 3, Ref. 36).

GENOMIC AND EPIGENOMIC PREDICTORS FOR VARIOUS CLINICAL PHENOTYPES OF MYASTHENIA GRAVIS

The aim: To evaluate the relationship of certain alleles of HLA class II leukocyte antigens and the profile of antibodies to various subunits of nicotinic acetylcholine receptors (nAChR), the level of Treg lymphocytes and the serum concentration of anti-inflammatory IL-10 for various clinical myasthenia gravis phenotypes. Materials and methods: We examined 217 patients with thymus-independent myasthenia (n = 42) and thymus-dependent myasthenia, among them patients with thymus hyperplasia (n = 108) and thymoma (n = 67). We used the following methods: ELISA, flow cytometry, light and fluorescence microscopy. Results: Certain genomic (polymorphism of leukocyte HLA-DR antigens) and epigenomic (antibodies to α1 and α7 nAChR subunits, expression of Treg lymphocytes and concentration of cytokines) predictors were identified for various myasthenia phenotypes. The presence of HLA haplotypes DR2 and DR7 in some young patients with M with disease progression led to the development of myasthenia gravis with thymoma (MT) at an older age. The presence of α7 nAChR subunit on thymocyte mitochondria was revealed, which is an additional autoimmune target for autoantibodies in patients with myasthenia gravis. An increase in the concentration of cytokines (IL-4, IL-8, IFN-γ) in all patients with myasthenia gravis was revealed. Conclusions: Estimate the features of the formation of various variants of the immune response in thymus-independent and thymus-dependent myasthenia gravis is a necessary condition for targeted immunocorrection or surgery.

A pain killer without analgesic tolerance designed by co-targeting PSD-95-nNOS interaction and α2-containning GABAARs.

Overactivation of N-methyl-D-aspartate receptor (NMDAR) in the spinal cord dorsal horn (SDH) in the setting of injury represents a key mechanism of neuropathic pain. However, directly blocking NMDAR or its downstream signaling, interaction between postsynaptic density-95 (PSD-95) and neuronal nitric oxide synthase (nNOS), causes analgesic tolerance, mainly due to GABAergic disinhibition. The aim of this study is to explore the possibility of preventing analgesic tolerance through co-targeting NMDAR downstream signaling and γ-aminobutyric acid type A receptors (GABAARs).Methods: Mechanical/thermal hyperalgesia were quantified to assess analgesic effects. Miniature postsynaptic currents were tested by patch-clamp recording to evaluate synaptic transmission in the SDH. GABA-evoked currents were tested on HEK293 cells expressing different subtypes of recombinant GABAARs to assess the selectivity of (+)-borneol and ZL006-05. The expression of α2 and α3 subunits of GABAARs and BDNF, and nNOS-PSD-95 complex levels were analyzed by western blotting and coimmunoprecipitation respectively. Open field test, rotarod test and Morris water maze task were conducted to evaluate the side-effect of ZL006-05.Results: (+)-Borneol selectively potentiated α2- and α3-containing GABAARs and prevented the disinhibition of laminae I excitatory neurons in the SDH and analgesic tolerance caused by chronic use of ZL006, a nNOS-PSD-95 blocker. A dual-target compound ZL006-05 produced by linking ZL006 and (+)-borneol through an ester bond blocked nNOS-PSD-95 interaction and potentiated α2-containing GABAAR selectively. Chronic use of ZL006-05 did not produce analgesic tolerance and unwanted side effects.Conclusion: By targeting nNOS-PSD-95 interaction and α2-containing GABAAR simultaneously, chronic use of ZL006-05 can avoid analgesic tolerance and unwanted side effects. Therefore, we offer a novel candidate drug without analgesic tolerance for treating neuropathic pain.

Collagen IVα345 dysfunction in glomerular basement membrane diseases. I. Discovery of a COL4A3 variant in familial Goodpasture’s and Alport diseases

Diseases of the glomerular basement membrane (GBM), such as Goodpasture’s disease (GP) and Alport syndrome (AS), are a major cause of chronic kidney failure and an unmet medical need. Collagen IVα345 is an important architectural element of the GBM that was discovered in previous research on GP and AS. How this collagen enables GBM to function as a permselective filter and how structural defects cause renal failure remain an enigma. We found a distinctive genetic variant of collagen IVα345 in both a familial GP case and four AS kindreds that provided insights into these mechanisms. The variant is an 8-residue appendage at the C-terminus of the α3 subunit of the α345 hexamer. A knock-in mouse harboring the variant displayed GBM abnormalities and proteinuria. This pathology phenocopied AS, which pinpointed the α345 hexamer as a focal point in GBM function and dysfunction. Crystallography and assembly studies revealed underlying hexamer mechanisms, as described in Boudko et al. and Pedchenko et al. Bioactive sites on the hexamer surface were identified where pathogenic pathways of GP and AS converge and, potentially, that of diabetic nephropathy (DN). We conclude that the hexamer functions include signaling and organizing macromolecular complexes, which enable GBM assembly and function. Therapeutic modulation or replacement of α345 hexamer could therefore be a potential treatment for GBM diseases, and this knock-in mouse model is suitable for developing gene therapies.

The α3 subunit of GABAA receptors promotes formation of inhibitory synapses in the absence of collybistin

Signaling at nerve cell synapses is a key determinant of proper brain function, and synaptic defects—or synaptopathies—are at the basis of many neurological and psychiatric disorders. Collybistin (CB), a brain-specific guanine nucleotide exchange factor, is essential for the formation of γ-aminobutyric acidergic (GABAergic) postsynapses in defined regions of the mammalian forebrain, including the hippocampus and basolateral amygdala. This process depends on a direct interaction of CB with the scaffolding protein gephyrin, which leads to the redistribution of gephyrin into submembranous clusters at nascent inhibitory synapses. Strikingly, synaptic clustering of gephyrin and GABAA type A receptors (GABAARs) in several brain regions, including the cerebral cortex and certain thalamic areas, is unperturbed in CB-deficient mice, indicating that the formation of a substantial subset of inhibitory postsynapses must be controlled by gephyrin-interacting proteins other than CB. Previous studies indicated that the α3 subunit of GABAARs (GABAAR-α3) binds directly and with high affinity to gephyrin. Here, we provide evidence (i) that a homooligomeric GABAAR-α3A343W mutant induces the formation of submembranous gephyrin clusters independently of CB in COS-7 cells, (ii) that gephyrin clustering is unaltered in the neuronal subpopulations endogenously expressing the GABAAR-α3 in CB-deficient brains, and (iii) that exogenous expression of GABAAR-α3 partially rescues impaired gephyrin clustering in CB-deficient hippocampal neurons. Our results identify an important role of GABAAR-α3 in promoting gephyrin-mediated and CB-independent formation of inhibitory postsynapses.

토끼 방향특이성 망막 신경절세포의 니코티닉 아세틸콜린 수용체 α7과 β4 소단위의 위치 측정

토끼 방향특이성 망막 신경절세포의 니코티닉 아세틸콜린 수용체 α7과 β4 소단위의 위치 측정

Identification of intraneuronal amyloid beta oligomers in locus coeruleus neurons of Alzheimer's patients and their potential impact on inhibitory neurotransmitter receptors and neuronal excitability.

Amyloid β-oligomers (AβO) are potent modulators of Alzheimer's pathology, yet their impact on one of the earliest brain regions to exhibit signs of the condition, the locus coeruleus (LC), remains to be determined. Of particular importance is whether AβO impact the spontaneous excitability of LC neurons. This parameter determines brain-wide noradrenaline (NA) release, and thus NA-mediated brain functions, including cognition, emotion and immune function, which are all compromised in Alzheimer's patients. Therefore, the aim of the study was to determine the expression profile of AβO in the LC of Alzheimer's patients and to probe their potential impact on the molecular and functional correlates of LC excitability, using a mouse model of increased Aβ production (APP-PSEN1). Immunohistochemistry and confocal microscopy, using AβO-specific antibodies, confirmed LC AβO expression both intraneuronally and extracellularly in both Alzheimer's and APP-PSEN1 samples. Patch clamp electrophysiology recordings revealed that APP-PSEN1 LC neuronal hyperexcitability accompanied this AβO expression profile, arising from a diminished inhibitory effect of GABA due to impaired expression and function of the GABA-A receptor (GABAA R) α3 subunit. This altered LC α3-GABAA R expression profile overlapped with AβO expression in samples from both APP-PSEN1 mice and Alzheimer's patients. Finally, strychnine-sensitive glycine receptors (GlyRs) remained resilient to Aβ-induced changes and their activation reversed LC hyperexcitability. The data suggest a close association between AβO and α3-GABAA Rs in the LC of Alzheimer's patients, and their potential to dysregulate LC activity, thereby contributing to the spectrum of pathology of the LC-NA system in this condition.

An inherent dysfunction in soluble guanylyl cyclase is present in the airway of severe asthmatics and is associated with aberrant redox enzyme expression and compromised NO-cGMP signaling

A subset of asthmatics develop a severe form of the disease whose etiology involves airway inflammation along with inherent drivers that remain ill-defined. To address this, we studied human airway smooth muscle cells (HASMC), whose relaxation drives airway bronchodilation and whose dysfunction contributes to airway obstruction and hypersensitivity in severe asthma. Because HASMC relaxation can be driven by the NO-soluble guanylyl cyclase (sGC)-cGMP signaling pathway, we questioned if HASMC from severe asthma donors might possess inherent defects in their sGC or in redox enzymes that support sGC function. We analyzed HASMC primary lines derived from 17 severe asthma and 16 normal donors and corresponding lung tissue samples regarding sGC activation by NO or by pharmacologic agonists, and also determined expression levels of sGC α1 and β1 subunits, supporting redox enzymes, and related proteins. We found a majority of the severe asthma donor HASMC (12/17) and lung samples primarily expressed a dysfunctional sGC that was NO-unresponsive and had low heterodimer content and high Hsp90 association. This sGC phenotype correlated with lower expression levels of the supporting redox enzymes cytochrome b5 reductase, catalase, and thioredoxin-1, and higher expression of heme oxygenases 1 and 2. Together, our work reveals that severe asthmatics are predisposed toward defective NO-sGC-cGMP signaling in their airway smooth muscle due to an inherent sGC dysfunction, which in turn is associated with inherent changes in the cell redox enzymes that impact sGC maturation and function.

The Vacuolar H+ ATPase α3 Subunit Negatively Regulates Migration and Invasion of Human Pancreatic Ductal Adenocarcinoma Cells

The Vacuolar H+ ATPase α3 Subunit Negatively Regulates Migration and Invasion of Human Pancreatic Ductal Adenocarcinoma Cells

Identification of the Aedes aegypti nAChR gene family and molecular target of spinosad.

Spinosad is an insecticide with unique mode of action (MOA) of disrupting nicotinic acetylcholine receptor and is efficacious against many insect species. Mutations in the nicotinic acetylcholine receptor (nAChR) α6 subunit have been identified that are associated with levels of spinosad resistance, but the molecular characterization of the nAChR gene family and a causative association between nAChR α6 and resistance to spinosad in Aedes aegypti, a primary vector of many arboviruses, have not yet been reported. In this study, we identified 10 candidate nAChR subunits in Ae. Aegypti, nAChRα1-α9 and nAChRβ1, showing similarly orthologous relationships with Anopheles gambiae. With the application of the CRISPR/Cas9 genome editing system, we introduced a 32-bp deletion at the 5' end of the Aaeα6 (Ae. aegypti nAChR α6) gene in a homozygous mutant strain (Aaeα6-KO). The mutation produced two successive pre-mature stop codons, resulting in loss of function in the target receptor. The Aaeα6-KO mutant strain exhibited a 320-fold level of resistance to spinosad compared with wildtype. A recessive mode of inheritance for spinosad resistance was found in the Aaeα6-KO strain. CRISPR/Cas9 introduced truncated Aaeα6 receptor in Ae. aegypti resulted in an increased level of resistance to spinosad, suggesting that the conserved nAChR α6 subunit is the target for spinosad insecticide. © 2020 Society of Chemical Industry.

GABAA Receptors Expressed in Oligodendrocytes Cultured from the Neonatal Rat Contain α3 and γ1 Subunits and Present Differential Functional and Pharmacological Properties.

Oligodendrocytes (OLs) express functional GABAA receptors (GABAARs) that are activated by GABA released at synaptic contacts with axons or by ambient GABA in extrasynaptic domains. In both instances, the receptors' molecular identity has not been fully defined. Furthermore, data on their structural diversity in different brain regions and information on age-dependent changes in their molecular composition are scant. This lack of knowledge has delayed access to a better understanding of the role of GABAergic signaling between neurons and OLs. Here, we used functional, and pharmacological analyses, as well as gene and protein expression of GABAAR subunits, to explore the subunit combination that could explain the receptor functional profile expressed in OLs from the neonate rat. We found that GABAAR composed of α3β2γ1 subunits mimicked the characteristics of the endogenous receptor when expressed heterologously in Xenopus laevis oocytes. Either α3 or γ1 subunit silencing by small interfering RNA transfection changed the GABA-response characteristics in oligodendrocyte precursor cells, indicating their participation in the endogenous receptor conformation. Thus, α3 subunit silencing shifted the mean EC50 for GABA from 75.1 to 46.6 µM, whereas γ1 silencing reduced the current amplitude response by 55%. We also observed that β-carbolines differentially enhance GABA responses in oligodendroglia as compared with those in neurons. These results contribute to defining the molecular and pharmacological properties of GABAARs in OLs. Additionally, the identification of β-carbolines as selective enhancers of GABAARs in OLs may help to study the role of GABAergic signaling during myelination. SIGNIFICANCE STATEMENT: GABAergic signaling through GABAA receptors (GABAARs) expressed in the oligodendroglial lineage contributes to the myelination control. Determining the molecular identity and the pharmacology of these receptors is essential to define their specific roles in myelination. Using GABAAR subunit expression and silencing, we identified that the GABAAR subunit combination α3β2γ1 conforms the bulk of GABAARs in oligodendrocytes from rat neonates. Furthermore, we found that these receptors have differential pharmacological properties that allow specific positive modulation by β-carbolines.

Expression of nAChRα7 receptor in model rats with Parkinson’s disease dementia

To investigate the expression and role of nicotinic acetylcholine α7 subunit (nAChRα7) in the hippocampus of rats with Parkinson's disease (PD) and Parkinson's disease dementia (PDD). PD model was established with 6-hydroxydopa. Then, methyllycaconitine was further used to prepare a PDD model. The behavioral indexes and malondialdehyde (MDA) and superoxide dismutase (SOD) levels were measured. The mRNA level and protein level of nAChRα7 in hippocampus was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western-blot, respectively. The α7nAchR and NeuN protein was detected by immunofluorescence. Compared with the PD model group, the escape latency of the PDD model group was significantly prolonged while the times of crossing platforms was significantly reduced (p < 0.05). The SOD level in the PDD and PD group decreased as compared with the control group (p < 0.05), while the MDA content increased (p < 0.05). The mRNA level and protein expression of nAChRα7 in the PDD and PD model group was significantly lower than that in the control group (p < 0.05). There was a significant difference in the nAChRα7 protein level between the PDD and PD model group (p < 0.05). Together, the nAChRα7 receptor level was significantly decreased in the PDD model group, indicating that the decrease in the nAChRα7 receptor levels is a possible mechanism of the pathogenesis of PDD.