Α2 Isoform Research Articles

Effect of Fe3+ on Na,K-ATPase: Unexpected activation of ATP hydrolysis.

Iron is a key element in cell function; however, its excess in iron overload conditions can be harmful through the generation of reactive oxygen species (ROS) and cell oxidative stress. Activity of Na,K-ATPase has been shown to be implicated in cellular iron uptake and iron modulates the Na,K-ATPase function from different tissues. In this study, we determined the effect of iron overload on Na,K-ATPase activity and established the role that isoforms and conformational states of this enzyme has on this effect. Total blood and membrane preparations from erythrocytes (ghost cells), as well as pig kidney and rat brain cortex, and enterocytes cells (Caco-2) were used. In E1-related subconformations, an enzyme activation effect by iron was observed, and in the E2-related subconformations enzyme inhibition was observed. The enzyme's kinetic parameters were significantly changed only in the Na+ curve in ghost cells. In contrast to Na,K-ATPase α2 and α3 isoforms, activation was not observed for the α1 isoform. In Caco-2 cells, which only contain Na,K-ATPase α1 isoform, the FeCl3 increased the intracellular storage of iron, catalase activity, the production of H2O2 and the expression levels of the α1 isoform. In contrast, iron did not affect lipid peroxidation, GSH content, superoxide dismutase and Na,K-ATPase activities. These results suggest that iron itself modulates Na,K-ATPase and that one or more E1-related subconformations seems to be determinant for the sensitivity of iron modulation through a mechanism in which the involvement of the Na, K-ATPase α3 isoform needs to be further investigated.

Na/K-ATPase Gene Expression in the Human Cochlea: A Study Using mRNA in situ Hybridization and Super-Resolution Structured Illumination Microscopy.

BackgroundThe pervasive Na/K-ATPase pump is highly expressed in the human cochlea and is involved in the generation of the endocochlear potential as well as auditory nerve signaling and relay. Its distribution, molecular organization and gene regulation are essential to establish to better understand inner ear function and disease. Here, we analyzed the expression and distribution of the ATP1A1, ATP1B1, and ATP1A3 gene transcripts encoding the Na/K-ATPase α1, α3, and β1 isoforms in different domains of the human cochlea using RNA in situ hybridization.Materials and MethodsArchival paraformaldehyde-fixed sections derived from surgically obtained human cochleae were used to label single mRNA gene transcripts using the highly sensitive multiplex RNAscope® technique. Localization of gene transcripts was performed by super-resolution structured illumination microscopy (SR-SIM) using fluorescent-tagged probes. GJB6 encoding of the protein connexin30 served as an additional control.ResultsSingle mRNA gene transcripts were seen as brightly stained puncta. Positive and negative controls verified the specificity of the labeling. ATP1A1 and ATP1B1 gene transcripts were demonstrated in the organ of Corti, including the hair and supporting cells. In the stria vascularis, these transcripts were solely expressed in the marginal cells. A large number of ATP1B1 gene transcripts were found in the spiral ganglion cell soma, outer sulcus, root cells, and type II fibrocytes. The ATP1B1 and ATP1A3 gene transcripts were rarely detected in axons.DiscussionSurgically obtained inner ear tissue can be used to identify single mRNA gene transcripts using high-resolution fluorescence microscopy after prompt formaldehyde fixation and chelate decalcification. A large number of Na/K-ATPase gene transcripts were localized in selected areas of the cochlear wall epithelium, fibrocyte networks, and spiral ganglion, confirming the enzyme’s essential role for human cochlear function.

Gold nanoclusters as elemental label for the sequential quantification of apolipoprotein E and metallothionein 2A in individual human cells of the retinal pigment epithelium using single cell-ICP-MS

Gold nanoclusters (AuNCs) with a diameter of 1.99 nm on average were synthesized and applied as labels in immunoprobes for the determination of cytosolic proteins in individual human retinal pigment epithelium (HRPEsv) cells by single cell – inductively coupled plasma – mass spectrometry (sc-ICP-MS). For quantitative purposes, the number of gold atoms per immunoprobe (i.e., the amplification factor) was determined; 466 gold atoms on average were obtained. Human metallothioneins (MT), including the 2A isoform (MT2A), and apolipoprotein E (APOE) play an important role under inflammation and oxidation processes in the RPE. The new single biomarker strategy introduced was applied to the sequential determination of MT2A and APOE in HRPEsv cells under pro-inflammatory and control conditions through the development of immunoassays with the corresponding AuNCs immunoprobes and the measurement of the 197Au+ signal by sc-ICP-MS. In addition, 56Fe+ signal was measured as constituent element of HRPEsv cells in order to check the integrity of the cells after the immunoassay and to confirm the number of cell events detected when monitoring the protein label (197Au+). Optimisation of parameters related with the sample preparation for the analysis of cytosolic proteins in intact HRPEsv cells was carried out. The method was successfully applied to the determination of both proteins in control cells and cells treated with the recombinant human interleukin-1α. Quantitative results obtained per cell for the average protein amounts of APOE and MT2A using the sc-ICP-MS procedure were corroborated with commercial ELISA kits.

Harmine suppresses collagen production in hepatic stellate cells by inhibiting DYRK1B

Liver fibrosis is a major consequence of chronic liver disease, where excess extracellular matrix is deposited, due caused by the activation of hepatic stellate cells (HSCs). The suppression of collagen production in HSCs is therefore regarded as a therapeutic target of liver fibrosis. The present study investigated effects of harmine, which is a β-carboline alkaloid and known as an inhibitor of dual-specificity tyrosine-regulated kinases (DYRKs), on the production of collagen in HSCs. LX-2 cells, a human HSC cell line, were treated with harmine (0–10 μM) for 48 h in the presence or absence of TGF-β1 (5 ng/ml). The expression of collagen type I α1 (COL1A1) and DYRK isoforms was investigated by Western blotting, quantitative RT-PCR, or immunofluorescence. The influence of knockdown of each DYRK isoform on the COL1A1 expression was further investigated. The expression of COL1A1 was markedly increased by treating with TGF-β1 for 48 h in LX-2 cells. Harmine (10 μM) significantly inhibited the increased expression of COL1A1. LX-2 cells expressed mRNAs of DYRK1A, DYRK1B, DYRK2, and DYRK4, although the expression of DYRK4 was much lower than the others. Knockdown of DYRK1B, but not DYRK1A or DYRK2, with siRNA significantly suppressed TGF-β1-induced increase in COL1A1 expression. These results suggest that harmine suppresses COL1A1 expression via inhibiting DYRK1B in HSCs and therefore might be effective for the treatment of liver fibrosis.

Spectroscopic observations of β-eudesmol binding to human cytochrome P450 isoforms 3A4 and 1A2, but not to isoforms 2C9, 2C19, and 2D6

β-Eudesmol (BEU) is a sesquiterpenoid component of Atractylodes lancea with cytotoxic activity against cholangiocarcinoma. Its lipophilic nature makes BEU a likely substrate of human cytochrome P450 (P450) enzymes. Using ligand-binding difference spectroscopy, the affinities of this compound to recombinant CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 were investigated in Escherichia coli membrane preparations. CYP3A4 showed a type I spectral change, with a binding constant Ks of 77 ± 23 (mean ± SD) μM at 0.5 μM P450 (Ks/[P450] ≈ 155). The reference substrate testosterone (TES) and the inhibitor fluconazole bound to the enzyme with apparent affinities of 86 ± 4 μM (type I) and 21 μM (type II), respectively. BEU was bound by CYP3A4 in a non-cooperative manner (Hill coefficient n ≈ 0.8). CYP1A2 showed reverse type I difference spectra with either BEU or caffeine (CAF). The CYP1A2 affinity for BEU was higher (0.23 mM) than for CAF (0.37 mM) but lower than for phenacetin (0.11 mM, type I). BEU did not bind significantly to CYP2C9, CYP2C19, and CYP2D6. Confirmation of metabolic activity and studies on the involvement of other human P450 isoforms are required. Double-beam spectrometry is needed to validate Ks measurements made with a microplate reader.

Effects of Aspirin on the Bioavailability and Pharmacokinetics of Repaglinide: Possible Role of Cytochrome P450 3A Isoforms Inhibition by Aspirin

Effects of Aspirin on the Bioavailability and Pharmacokinetics of Repaglinide: Possible Role of Cytochrome P450 3A Isoforms Inhibition by Aspirin

Concentrations and Toxic Implications of Dioxin-Like Polychlorinated Biphenyls in Soil Samples from Electrical Power Stations in Lagos, Nigeria.

Dioxin-like polychlorinated biphenyls (dl-PCBs) are ubiquitous chemicals which mediate toxicity in a way similar to polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans. In silico modeling was used to predict the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of eight dioxin-like PCBs in soil samples of 12 power stations in Lagos, Nigeria. Concentrations of Σdl-PCB8 in soil samples ranged from 490 to 61,000 pg g-1 , with mean concentrations of 17,000 pg g-1 . The corresponding toxic equivalent (TEQ) concentrations of Ʃdl-PCB8 ranged from 0.01 to 450 pg TEQ g-1 , with a mean value of 42 pg TEQ g-1 . Mean TEQ concentrations for Ʃdl-PCB8 in soil samples from all but one of the sites exceeded the Canadian guideline value of 4 pg TEQ g-1 and the US and German guideline values of 5-10 pg TEQ g-1 . However, the TEQ concentrations obtained were all below the US action level of 1000 pg TEQ g-1 . The ADMET predictions revealed that all studied dl-PCBs are inhibitors of three major isoforms (1A2, 2C9, and 2C19) of cytochrome P450 enzyme. Acute oral toxicity (median lethal dose) predictions revealed that all target dl-PCBs were class III compounds. Hepatotoxicity and carcinogenicity were positive, signifying that the studied compounds all have a tendency to elicit these effects. Occupational daily TEQ exposure via soil ingestion was estimated for an average adult worker weighing 70 kg. The maximum exposure obtained was 0.14 pg TEQ kg-1 body weight day-1 , which is half of the European Food Safety Authority (EFSA) tolerable daily intake (TDI) for dioxin-like compounds. This raises concern over the possible exceedance of the EFSA TDI for these workers if other dietary and nondietary exposure pathways and dioxin-like compounds are considered. Environ Toxicol Chem 2022;41:800-809. © 2021 SETAC.

Separation of intact proteins by capillary electrophoresis.

This work introduces novel and universal workflows for the analysis of intact proteins by capillary electrophoresis and presents guidelines for the targeted selection of appropriate background electrolytes (BGEs) by consideration of the target proteins' isoelectric point (pI). The suitability of neutral dimethyl polysiloxane (PDMS) capillaries with dynamic coatings of cationic cetyltrimethylammonium bromide (CTAB) or anionic sodium dodecyl sulfate (SDS), and bare fused silica (BFS) capillaries were systematically evaluated for the analysis of histidine and seven model proteins in six BGEs with pH values between 3.0 and 9.6. Multiple capillary and BGE combinations were suitable for the analysis of all proteins with molecular weights ranging from 13.7-150 kDa, and pIs between 4.7 and 9.6. The CTAB-PDMS capillary was best suited for low pH BGEs, while the SDS-PDMS and BFS capillary were superior for high pH BGEs. These combinations consistently resulted in sharp peak shapes and rapid migration times. pH values of BGEs closer to the proteins' pI produced poorer peak shapes and decreased effective mobilities due to suppressed ionisation. Plots of mobility vs. pH crossed at approximately the pI of the protein in most cases. The workflow was applied to the analysis of caseins and whey proteins in milk for the separation of the seven most abundant proteins, including the isoforms of A1 and A2 β-casein and β-lactoglobulin A and B.

Association of FSHR and DENND1A polymorphisms with polycystic ovary syndrome: a meta-analysis.

Multiple genetic variants have been studied for years to try to find an association with polycystic ovary syndrome (PCOS). This meta-analysis will investigate if there are associations between increased risk of PCOS and rs6165 polymorphism in follicle stimulating hormone receptor (FSHR) gene and rs2479106 polymorphism in differentially expressed in Differentially Expressed in Normal and Neoplastic Development Isoform 1A (DENND1A) gene. Studies were identified from PubMed library, and case-control studies with correct polymorphisms and available genotype frequencies were included. The statistical analysis is done in Review Manager 5.3, and odds ratio (OR) with corresponding 95% confidence interval (CI) was calculated to see if any association with PCOS exists. In the study of FSHR gene, eight articles with 1539 cases and 1877 controls were included. No relations were found between PCOS and rs6165 polymorphism in neither the allelic (OR=1.07, 95% CI=0.97-1.19, p=0.18), recessive (OR=1.21, 95% CI=0.98-1.50, p=0.07) nor the dominant (OR=1.05, 95% CI=0.91-1.20, p=0.53) model. The rs2479106 polymorphism in DENND1A gene included 10 studies with 3627 cases and 20325 controls. Only the Asian subgroup in the recessive model (OR=1.84, 95% CI=1.19-2.85, p=0.006) showed a positive relation with PCOS, while associations were not found within the overall results in the allelic (OR=1.09, 95% CI=0.98-1.21, p=0.10), recessive (OR=1.26, 95% CI=0.73-2.19, p=0.41) or the dominant (OR=1.31, 95% CI=1.00-1.71, p=0.05) model. This meta-analysis suggests that rs2479106 polymorphism in DENND1A gene is associated with increased risk of PCOS in the Asian population. No relations were found with increased risk of PCOS and rs6165 polymorphism in FSHR gene.

ALDH1A3 Segregated Expression and Nucleus-Associated Proteasomal Degradation Are Common Traits of Glioblastoma Stem Cells.

Aldehyde dehydrogenase 1 isoforms A1 and A3 have been implicated as functional biomarkers associated with distinct molecular subtypes of glioblastoma and glioblastoma stem cells. However, the exact roles of these isoforms in different types of glioma cells remain unclear. The purpose of this study was to dissect the association of A1 or A3 isoforms with stem and non-stem glioblastoma cells. This study has undertaken a systematic characterization of A1 and A3 proteins in glioblastoma tissues and a panel of glioblastoma stem cells using immunocytochemical and immunofluorescence staining, Western blot and the subcellular fractionation methodology. Our main findings are (i) human GSCs express uniformly ALDH1A3 but not the ALDH1A1 isoform whereas non-stem glioma cells comparably express both isoforms; (ii) there is an abundance of ALDH1A3 peptides that prevail over the full-length form in glioblastoma stem cells but not in non-stem glioma cells; (iii) full-length ALDH1A3 and ALDH1A3 peptides are spatially segregated within the cell; and (vi) the abundance of full-length ALDH1A3 and ALDH1A3 peptides is sensitive to MG132-mediated proteasomal inhibition. Our study further supports the association of ALDH1A3 with glioblastoma stem cells and provide evidence for the regulation of ALDH1A3 activities at the level of protein turnover.

Effect of AMPKα2 knockdown on Alzheimer's pathogenesis and cognitive deficits in young Tg19959 mice.

AMP-activated protein kinase (AMPK) is a cellular energy censor that has been implicated in Alzheimer's disease (AD). The catalytic α isoforms (α1 and α2) of AMPK are normally expressed in a homeostatic equilibrium but are differentially expressed in AD brains, with α2 expression significantly decreased compared to controls. AMPK dysregulation may be an early feature of AD that promotes accelerated cognitive decline through its roles in protein synthesis regulation, metabolism, autophagy, and mitochondrial biogenesis. The early loss of the α2 isoform may exacerbate or accelerate disease progression in the preclinical stage. AMPKα2 was conditionally knocked down in excitatory forebrain neurons in the Tg19959 AD mouse model to create a heterozygous AMPKα2/Tg19959 double mutant. At 3-5 months of age, before cognitive impairments become evident, the mice were subjected to the Morris Water Maze (MWM) and Novel Object Recognition (NOR) task that measures hippocampus-dependent spatial learning and memory. In addition, Golgi staining, immunohistochemistry, and transmission electron microscopy of the hippocampus were performed. The double mutant AMPKα2/Tg19959 mice showed significant memory impairments in both the MWM and NOR task compared to wild type, Tg19959, and α2/Cre littermates. The AMPKα2/Tg19959 double mutant mice also showed significant decreases in post-synaptic density size and in the number of polyribosomes. Differences in spine morphology and amyloid plaque deposition were also observed. AMPKα2 knockdown in a "pre-symptomatic" AD mouse model leads to accelerated cognitive deficits and disruption of protein synthesis regulatory mechanisms. Taken together, these findings suggest that reduction of AMPKα2 may exacerbate or accelerate cognitive decline and AD pathogenesis. This finding could provide a future therapeutic target for the treatment of AD.

Gene expression changes related to bone mineralization, blood pressure and lipid metabolism in mouse kidneys after space travel

Space travel burdens health by imposing considerable environmental stress associated with radioactivity and microgravity. In particular, gravity change predominantly impacts blood pressure and bone homeostasis, both of which are controlled mainly by the kidneys. Nuclear factor erythroid-2-related transcription factor 2 (Nrf2) plays essential roles in protecting the kidneys from various environmental stresses and injuries. To elucidate the effects of space travel on mammals in preparation for the upcoming space era, our study investigated the contribution of Nrf2 to kidney function in mice two days after their return from a 31-day stay in the International Space Station using Nrf2 knockout mice. Meaningfully, expression levels of genes regulating bone mineralization, blood pressure and lipid metabolism were found to be significantly altered in the kidneys after space travel in an Nrf2-independent manner. In particular, uridine diphosphate-glucuronosyltransferase 1A (Ugt1a) isoform genes were found to be expressed in an Nrf2-dependent manner and induced exclusively in the kidneys after return to Earth. Since spaceflight elevated the concentrations of fatty acids in the mouse plasma, we suggest that Ugt1a isoform expression in the kidneys was induced to promote glucuronidation of excessively accumulated lipids and excrete them into urine after the return from space. Thus, the kidneys were proven to play central roles in adaptation to gravity changes caused by going to and returning from space by controlling blood pressure and bone mineralization. Additionally, kidney Ugt1a isoform induction after space travel implies a significant role of the kidneys for space travelers in the excretion of excessive lipids.

Variation in Expression of Cytochrome P450 3A Isoforms and Toxicological Effects: Endo- and Exogenous Substances as Regulatory Factors and Substrates.

The CYP3A subfamily, which includes isoforms CYP3A4, CYP3A5, and CYP3A7 in humans, plays important roles in the metabolism of various endogenous and exogenous substances. Gene and protein expression of CYP3A4, CYP3A5, and CYP3A7 show large inter-individual differences, which are caused by many endogenous and exogenous factors. Inter-individual differences can cause negative outcomes, such as adverse drug events and disease development. Therefore, it is important to understand the variations in CYP3A expression caused by endo- and exogenous factors, as well as the variation in the metabolism and kinetics of endo- and exogenous substrates. In this review, we summarize the factors regulating CYP3A expression, such as bile acids, hormones, microRNA, inflammatory cytokines, drugs, environmental chemicals, and dietary factors. In addition, variations in CYP3A expression under pathological conditions, such as coronavirus disease 2019 and liver diseases, are described as examples of the physiological effects of endogenous factors. We also summarize endogenous and exogenous substrates metabolized by CYP3A isoforms, such as cholesterol, bile acids, hormones, arachidonic acid, vitamin D, and drugs. The relationship between the changes in the kinetics of these substrates and the toxicological effects in our bodies are discussed. The usefulness of these substrates and metabolites as endogenous biomarkers for CYP3A activity is also discussed. Notably, we focused on discrimination between CYP3A4, CYP3A5, and CYP3A7 to understand inter-individual differences in CYP3A expression and function.

ICYP-MFE: Identifying Human Cytochrome P450 Inhibitors Using Multitask Learning and Molecular Fingerprint-Embedded Encoding.

The human cytochrome P450 (CYP) superfamily holds responsibilities for the metabolism of both endogenous and exogenous compounds such as drugs, cellular metabolites, and toxins. The inhibition exerted on the CYP enzymes is closely associated with adverse drug reactions encompassing metabolic failures and induced side effects. In modern drug discovery, identification of potential CYP inhibitors is, therefore, highly essential. Alongside experimental approaches, numerous computational models have been proposed to address this biochemical issue. In this study, we introduce iCYP-MFE, a computational framework for virtual screening on CYP inhibitors toward 1A2, 2C9, 2C19, 2D6, and 3A4 isoforms. iCYP-MFE contains a set of five robust, stable, and effective prediction models developed using multitask learning incorporated with molecular fingerprint-embedded features. The results show that multitask learning can remarkably leverage useful information from related tasks to promote global performance. Comparative analysis indicates that iCYP-MFE achieves three predominant tasks, one equivalent task, and one less effective task compared to state-of-the-art methods. The area under the receiver operating characteristic curve (AUC-ROC) and the area under the precision-recall curve (AUC-PR) were two decisive metrics used for model evaluation. The prediction task for CYP2D6-inhibition achieves the highest AUC-ROC value of 0.93 while the prediction task for CYP1A2-inhibition obtains the highest AUC-PR value of 0.92. The substructural analysis preliminarily explains the nature of the CYP-inhibitory activity of compounds. An online web server for iCYP-MFE with a user-friendly interface was also deployed to support scientific communities in identifying CYP inhibitors.

Are isoforms of capacitating Na+ K+ -ATPase localized to sperm head rafts?

Capacitation begins in the sperm head plasma membrane (HPM). Membrane rafts could house signaling molecules, but although these specialized microdomains have been microscopically visualized in sperm heads, rafts have been isolated for study only from homogenized whole sperm or tails, never purified HPM. Sodium/potassium ATPase (Na+ K+ -ATPase) is a membrane-bound signaling protein that induces capacitation in bull sperm in response to the steroid hormone ouabain, and its subunit isoforms α1, α3, β1, β2, and β3 are known in HPM. This study hypothesized that rafts exist in the HPM of bull sperm, with Na+ K+ -ATPase subunit isoforms preferentially localized there. Western immunoblotting (WB) of HPM from fresh, uncapacitated bull sperm (n = 7 ejaculates), and detergent-resistant membranes isolated by density gradient centrifugation from this HPM, contained the raft-marker protein Flotillin-1; the non-raft fraction did not. HPM, raft, and non-raft contained all known Na+ K+ -ATPase isoforms including, for the first time, the previously unknown α2 isoform. Quantification (ImageQuant Software) found α3 and β1 were relatively dominant isoforms in the HPM raft. WB profiles of raft isoforms differed significantly from HPM and non-raft profiles, with unique banding patterns and amounts, hinting that the capacitation signaling in the now-identified HPM rafts may depend on unique sequences within the isoform structure.

Cytochrome P450 isoforms 1A1, 1B1 AND 2W1 as targets for therapeutic intervention in head and neck cancer

Epidemiological studies have shown that head and neck cancer (HNC) is a complex multistage process that in part involves exposure to a combination of carcinogens and the capacity of certain drug-metabolising enzymes including cytochrome P450 (CYP) to detoxify or activate such carcinogens. In this study, CYP1A1, CYP1B1 and CYP2W1 expression in HNC was correlated with potential as target for duocarmycin prodrug activation and selective therapy. In the HNC cell lines, elevated expression was shown at the gene level for CYP1A1 and CYP1B1 whereas CYP2W1 was hardly detected. However, CYP2W1 was expressed in FaDu and Detroit-562 xenografts and in a cohort of human HNC samples. Functional activity was measured in Fadu and Detroit-562 cells using P450-Glo™ assay. Antiproliferative results of duocarmycin prodrugs ICT2700 and ICT2706 revealed FaDu and Detroit-562 as the most sensitive HNC cell lines. Administration of ICT2700 in vivo using a single dose of ICT2700 (150 mg/kg) showed preferential inhibition of small tumour growth (mean size of 60 mm3) in mice bearing FaDu xenografts. Significantly, our findings suggest a potential targeted therapeutic approach to manage HNCs by exploiting intratumoural CYP expression for metabolic activation of duocarmycin-based prodrugs such as ICT2700.

Comparative description of the mRNA expression profile of Na+ /K+ -ATPase isoforms in adult mouse nervous system.

Mutations in genes encoding Na+/K+‐ATPase α1, α2, and α3 subunits cause a wide range of disabling neurological disorders, and dysfunction of Na+/K+‐ATPase may contribute to neuronal injury in stroke and dementia. To better understand the pathogenesis of these diseases, it is important to determine the expression patterns of the different Na+/K+‐ATPase subunits within the brain and among specific cell types. Using two available scRNA‐Seq databases from the adult mouse nervous system, we examined the mRNA expression patterns of the different isoforms of the Na+/K+‐ATPase α, β and Fxyd subunits at the single‐cell level among brain regions and various neuronal populations. We subsequently identified specific types of neurons enriched with transcripts for α1 and α3 isoforms and elaborated how α3‐expressing neuronal populations govern cerebellar neuronal circuits. We further analyzed the co‐expression network for α1 and α3 isoforms, highlighting the genes that positively correlated with α1 and α3 expression. The top 10 genes for α1 were Chn2, Hpcal1, Nrgn, Neurod1, Selm, Kcnc1, Snrk, Snap25, Ckb and Ccndbp1 and for α3 were Sorcs3, Eml5, Neurod2, Ckb, Tbc1d4, Ptprz1, Pvrl1, Kirrel3, Pvalb, and Asic2.

Effect of handling on ATP utilization of cerebral Na,K-ATPase in rats with trimethyltin-induced neurodegeneration.

Previously it was shown that for reduction of anxiety and stress of experimental animals, preventive handling seems to be one of the most effective methods. The present study was oriented on Na,K-ATPase, a key enzyme for maintaining proper concentrations of intracellular sodium and potassium ions. Malfunction of this enzyme has an essential role in the development of neurodegenerative diseases. It is known that this enzyme requires approximately 50% of the energy available to the brain. Therefore in the present study utilization of the energy source ATP by Na,K-ATPase in the frontal cerebral cortex, using the method of enzyme kinetics was investigated. As a model of neurodegeneration treatment with trimethyltin (TMT) was applied. Daily handling (10min/day) of healthy rats and rats suffering neurodegeneration induced by administration of TMT in a dose of (7.5mg/kg), at postnatal days 60-102 altered the expression of catalytic subunits of Na,K-ATPase as well as kinetic properties of this enzyme in the frontal cerebral cortex of adult male Wistar rats. In addition to the previously published beneficial effect on spatial memory, daily treatment of rats was accompanied by improved maintenance of sodium homeostasis in the frontal cortex. The key system responsible for this process, Na,K-ATPase, was able to utilize better the energy substrate ATP. In rats, manipulation of TMT-induced neurodegeneration promoted the expression of the α2 isoform of the enzyme, which is typical for glial cells. In healthy rats, manipulation was followed by increased expression of the α3 subunit, which is typical of neurons.

Diseases caused by mutations in the Na+/K+ pump α1 gene ATP1A1.

Human cell survival requires function of the Na+/K+ pump; the heteromeric protein that hydrolyzes ATP to extrude Na+ and import K+ across the plasmalemma, thereby building and maintaining these ions' electrochemical gradients. Numerous dominant diseases caused by mutations in genes encoding for Na+/K+ pump catalytic (α) subunit isoforms highlight the importance of this protein. Here, we review literature describing disorders caused by missense mutations in ATP1A1, the gene encoding the ubiquitously expressed α1 isoform of the Na+/K+ pump. These various maladies include primary aldosteronism with secondary hypertension, an endocrine syndrome, Charcot-Marie-Tooth disease, a peripheral neuropathy, complex spastic paraplegia, another neuromuscular disorder, as well as hypomagnesemia accompanied by seizures and cognitive delay, a condition affecting the renal and central nervous systems. This article focuses on observed commonalities among these mutations' functional effects, as well as on the special characteristics that enable each particular mutation to exclusively affect a certain system, without affecting others. In this respect, it is clear how somatic mutations localized to adrenal adenomas increase aldosterone production without compromising other systems. However, it remains largely unknown how and why some but not all de novo germline or familial mutations (where the mutant must be expressed in numerous tissues) produce a specific disease and not the other diseases. We propose hypotheses to explain this observation and the approaches that we think will drive future research on these debilitating disorders to develop novel patient-specific treatments by combining the use of heterologous protein-expression systems, patient-derived pluripotent cells, and gene-edited cell and mouse models.

Genetic Basis of Type IV Collagen Disorders of the Kidney.

The glomerular basement membrane is a vital component of the filtration barrier of the kidney and is primarily composed of a highly structured matrix of type IV collagen. Specific isoforms of type IV collagen, the α3(IV), α4(IV), and α5(IV) isoforms, assemble into trimers that are required for normal glomerular basement membrane function. Disruption or alteration in these isoforms leads to breakdown of the glomerular basement membrane structure and function and can lead to progressive CKD known as Alport syndrome. However, there is wide variability in phenotype among patients with mutations affecting type IV collagen that depends on a complex interplay of sex, genotype, and X-chromosome inactivation. This article reviews the genetic basis of collagen disorders of the kidney as well as potential treatments for these conditions, including direct alteration of the DNA, RNA therapies, and manipulation of collagen proteins.