Alpha1 Subunit Research Articles

Biallelic variants in NOS3 and GUCY1A3, the two major genes of the nitric oxide pathway, cause moyamoya cerebral angiopathy

BackgroundMoyamoya angiopathy (MMA) is a rare cerebrovascular condition leading to stroke. Mutations in 15 genes have been identified in Mendelian forms of MMA, but they explain only a very small proportion of cases. Our aim was to investigate the genetic basis of MMA in consanguineous patients having unaffected parents in order to identify genes involved in autosomal recessive MMA.MethodsExome sequencing (ES) was performed in 6 consecutive consanguineous probands having MMA of unknown etiology. Functional consequences of variants were assessed using western blot and protein 3D structure analyses.ResultsCausative homozygous variants of NOS3, the gene encoding the endothelial nitric oxide synthase (eNOS), and GUCY1A3, the gene encoding the alpha1 subunit of the soluble guanylate cyclase (sGC) which is the major nitric oxide (NO) receptor in the vascular wall, were identified in 3 of the 6 probands. One NOS3 variant (c.1502 + 1G > C) involves a splice donor site causing a premature termination codon and leads to a total lack of eNOS in endothelial progenitor cells of the affected proband. The other NOS3 variant (c.1942 T > C) is a missense variant located into the flavodoxine reductase domain; it is predicted to be destabilizing and shown to be associated with a reduction of eNOS expression. The GUCY1A3 missense variant (c.1778G > A), located in the catalytic domain of the sGC, is predicted to disrupt the tridimensional structure of this domain and to lead to a loss of function of the enzyme. Both NOS3 mutated probands suffered from an infant-onset and severe MMA associated with posterior cerebral artery steno-occlusive lesions. The GUCY1A3 mutated proband presented an adult-onset MMA associated with an early-onset arterial hypertension and a stenosis of the superior mesenteric artery. None of the 3 probands had achalasia.ConclusionsWe show for the first time that biallelic loss of function variants in NOS3 is responsible for MMA and that mutations in NOS3 and GUCY1A3 are causing fifty per cent of MMA in consanguineous patients. These data pinpoint the essential role of the NO pathway in MMA pathophysiology.

A Role for the Proteasome Alpha2 Subunit N-Tail in Substrate Processing.

The proteolytic active sites of the 26S proteasome are sequestered within the catalytic chamber of its 20S core particle (CP). Access to this chamber is through a narrow channel defined by the seven outer α subunits. In the resting state, the N-termini of neighboring α subunits form a gate blocking access to the channel. The attachment of the activators or regulatory particles rearranges the blocking α subunit N-termini facilitating the entry of substrates. By truncating or mutating each of the participating α N-termini, we report that whereas only a few N-termini are important for maintaining the closed gate, all seven N-termini participate in the open gate. Specifically, the open state is stabilized by a hydrogen bond between an invariant tyrosine (Y) in each subunit with a conserved aspartate (D) in its counterclockwise neighbor. The lone exception is the α1-α2 pair leaving a gap in the ring circumference. The third residue (X) of this YD(X) motif aligns with the open channel. Phenylalanine at this position in the α2 subunit comes in direct contact with the translocating substrate. Consequently, deletion of the α2 N-terminal tail attenuates proteolysis despite the appearance of an open gate state. In summary, the interlacing N-terminal YD(X) motifs regulate both the gating and translocation of the substrate.

Molecular and Functional Changes to Postsynaptic Cholinergic Signaling in the Vestibular Sensory Organs of Aging C57BL/6Mice.

Cholinergic circuits in the central nervous system are vulnerable to age-related functional decline, but it is not known if aging impacts cholinergic signaling in the vestibular sensory organs, which are critically important to balance maintenance and visual gaze stability. We have previously shown cholinergic neurotransmission between vestibular efferent terminals and type II mechanosensory hair cells requires the alpha9 (Chrna9) nicotinic receptor subunit. Homozygous knockout of the alpha9 subunit causes vestibulo-ocular reflex adaptation deficits that mirror those observed in aged mice. This prompted examination of cholinergic signaling in the vestibular sensory organs of aged mice. We confirmed older (>24months) mice had impaired performance in a balance beam task compared to young (3-4months) adult mice. While there was no qualitative loss of cholinergic axon varicosities in the crista ampullaris of old mice, qPCR analysis revealed reduced expression of nicotinic receptor subunit genes Chrna1, Chrna9, and Chrna10 in the cristae of old relative to young mice. Functionally, single-cell patch clamp recordings taken from type II vestibular hair cells exposed to acetylcholine show reduced conductance through alpha9/10 subunit-containing nicotinic receptors in older mice, despite preserved passive membrane properties and voltage-activated conductances. These findings suggest that cholinergic signaling in the peripheral vestibular sensory organs is vulnerable to aging processes, manifesting in dynamic molecular and functional age-related changes. Given the importance of these organs to our everyday activities, and the dramatic increase in fall incidence in the older, further investigation into the mechanisms of altered peripheral vestibular function in older humans is warranted.

Molecular changes underlying decay of sensory responses and enhanced seizure propensity in peritumoral neurons.

Glioblastoma growth impacts on the structure and physiology of peritumoral neuronal networks, altering the activity of pyramidal neurons which drives further tumor progression. It is therefore of paramount importance to identify glioma-induced changes in pyramidal neurons, since they represent a key therapeutic target. We longitudinal monitored visual evoked potentials after the orthotopic implant of murine glioma cells into the mouse occipital cortex. With laser microdissection, we analyzed layer II-III pyramidal neurons molecular profile and with local field potentials recordings we evaluated the propensity to seizures in glioma-bearing animals with respect to control mice. We determine the time course of neuronal dysfunction of glioma-bearing mice and we identify a symptomatic stage, based on the decay of visual response. At that time point, we microdissect layer II-III pyramidal neurons and evaluate the expression of a panel of genes involved in synaptic transmission and neuronal excitability. Compared to the control group, peritumoral neurons show a decrease in the expression of the SNARE complex gene SNAP25 and the alpha1 subunit of the GABA-A receptor. No significant changes are detected in glutamatergic (ie, AMPA or NMDA receptor subunit) markers. Further reduction of GABA-A signaling by delivery of a benzodiazepine inverse agonist, DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) precipitates seizures in 2 mouse models of tumor-bearing mice. These studies reveal novel molecular changes that occur in the principal cells of the tumor-adjacent zone. These modifications may be therapeutically targeted to ameliorate patients' quality of life.

The Alpha-1 Subunit of the Na+/K+-ATPase (ATP1A1) Is a Host Factor Involved in the Attachment of Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea (PED) is an acute and severe atrophic enteritis caused by porcine epidemic diarrhea virus (PEDV) that infects pigs and makes huge economic losses to the global swine industry. Previously, researchers have believed that porcine aminopeptidase-N (pAPN) was the primary receptor for PEDV, but it has been found that PEDV can infect pAPN knockout pigs. Currently, the functional receptor for PEDV remains unspecified. In the present study, we performed virus overlay protein binding assay (VOPBA), found that ATP1A1 was the highest scoring protein in the mass spectrometry results, and confirmed that the CT structural domain of ATP1A1 interacts with PEDV S1. First, we investigated the effect of ATP1A1 on PEDV replication. Inhibition of hosts ATP1A1 protein expression using small interfering RNA (siRNAs) significantly reduced the cells susceptibility to PEDV. The ATP1A1-specific inhibitors Ouabain (a cardiac steroid) and PST2238 (a digitalis toxin derivative), which specifically bind ATP1A1, could block the ATP1A1 protein internalization and degradation, and consequently reduce the infection rate of host cells by PEDV significantly. Additionally, as expected, overexpression of ATP1A1 notably enhanced PEDV infection. Next, we observed that PEDV infection of target cells resulted in upregulation of ATP1A1 at the mRNA and protein levels. Furthermore, we found that the host protein ATP1A1 was involved in PEDV attachment and co-localized with PEDV S1 protein in the early stage of infection. In addition, pretreatment of IPEC-J2 and Vero-E6 cells with ATP1A1 mAb significantly reduced PEDV attachment. Our observations provided a perspective on identifying key factors in PEDV infection, and may provide valuable targets for PEDV infection, PEDV functional receptor, related pathogenesis, and the development of new antiviral drugs.

Altered Expression of Several Molecular Mediators of Cerebrospinal Fluid Production in Hyp Mice.

X-linked hypophosphatemia (XLH) is a genetic disease, causing life-long hypophosphatemia due to overproduction of fibroblast growth factor 23 (FGF23). XLH is associated with Chiari malformations, cranial synostosis, and syringomyelia. FGF23 signals through FGFR1c and requires a coreceptor, α-Klotho, which is expressed in the renal distal convoluted tubules and the choroid plexus (ChP). In the ChP, α-Klotho participates in regulating cerebrospinal fluid (CSF) production by shuttling the sodium/potassium adenosine triphosphatase (Na+/K+-ATPase) to the luminal membrane. The sodium/potassium/chloride cotransporter 1 (NKCC1) also makes a substantial contribution to CSF production. Since CSF production has not been studied in XLH, we sought to determine if there are changes in the expression of these molecules in the ChP of Hyp mice, the murine model of XLH, as a first step toward testing the hypothesis that altered CSF production contributes to the cranial and spinal malformations seen this disease. Semi-quantitative real-time PCR was used to analyze the level of expression of transcripts for Fgfr1c, and thee key regulators of CSF production, Klotho, Atp1a1 and Slc12a2. In situ hybridization was used to provide anatomical localization for the encoded proteins. Real-time polymerase chain reaction (RT-PCR) demonstrated significant upregulation of Klotho transcripts in the fourth ventricle of Hyp mice compared to controls. Transcript levels for Fgfr1c were unchanged in Hyp mice. Atp1a1 transcripts encoding the alpha-1 subunit of Na+/K+-ATPase were significantly downregulated in the third and lateral ventricles (LV). Expression levels of the Slc12a2 transcript (which encodes NKCC1) were unchanged in Hyp mice compared to controls. In situ hybridization (ISH) confirmed the presence of all 4 transcripts in the LV ChP both of WT and Hyp mice. This is the first study to document a significant change in the level of expression of the molecular machinery required for CSF production in Hyp mice. Whether similar changes occur in patients with XLH, potentially contributing to the cranial and spinal cord abnormalities frequently seen in XLH, remains to be determined.

GABAA Receptor and Serotonin Transporter Expression Changes Dissociate Following Mild Traumatic Brain Injury: Influence of Sex and Estrus Cycle Phase in Rats

Mild traumatic brain injuries (mild TBIs) can affect both males and females, but females are more likely to report long-term psychological complications, including changes in mood and generalized anxiety. Additionally, reproductive cycle phase has been shown to affect mild TBI symptom expression within females. These variances may result from sex differences in mild TBI-induced alterations to neurotransmission in brain regions that influence mood and emotion, possibly mediated by sex steroids. The hippocampus and amygdala are implicated in stress responses and anxiety, and within these regions, gamma-aminobutyric acid (GABA) and serotonin modulate output and behavioral expression. Metabolites of progesterone can allosterically enhance GABAergic signaling, and sex steroids are suggested to regulate the expression of the serotonin transporter (SERT). To determine how mild TBI might alter GABA receptor and SERT expression in males and females, immunocytochemistry was used to quantify expression of the alpha-1 subunit of the GABAA receptor (α1-GABAA), SERT, and a neuronal marker (NeuN) in the brains of adult male and naturally-cycling female rats, both with and without mild TBI, 17 days after injury. Mild TBI altered the expression of α1-GABAA in the amygdala and hippocampus in both sexes, but the direction of change observed depended on sex and reproductive cycle phase. In contrast, mild TBI had little effect on SERT expression. However, SERT expression differed between sexes and varied with the cycle phase. These findings demonstrate that regulation of neurotransmission following mild TBI differs between males and females, with implications for behavioral outcomes and the efficacy of therapeutic strategies.

Physiologic Functions and Therapeutic Applications of α7 Nicotinic Acetylcholine Receptor in Brain Disorders.

Accumulating data suggest that α7 nicotinic acetylcholine receptors (α7nAChRs) are an important therapeutic target for the treatment of Alzheimer's disease (AD) and schizophrenia. The homopentameric ligand-gated ion channel α7nAChR consists of five identical α7 subunits that are encoded by the CHRNA7 (cholinergic receptor nicotinic alpha7 subunit) gene. Moreover, α7nAChRs are densely distributed throughout the hippocampus, cortex, and thalamus brain regions, but sparsely in the striatum, forebrain, and medulla. Compared with other nAChRs, α7nAChR binds with low affinity to the naturally occurring neurotransmitter acetylcholine and the non-specific exogenous agonist nicotine, and with high affinity to the specific antagonists α-bungarotoxin and methyllycaconitine. Reports indicate that α7nAChR plays important roles in neurotransmitter release, cognitive functioning, and the cholinergic anti-inflammatory response. Genetic variations that alter CHRNA7 mRNA and protein expression or cause α7nAChR dysfunction are associated with many brain disorders. Our previous studies revealed that α7nAChR exerts neuroprotection in AD by acting as a cargo receptor for binding the autophagosomal marker protein LC3 and engulfing extracellular neurotoxic Aβ1-42 during autophagic degradation of the α7nAChR-Aβ1-42 complex. However, the role of α7nAChRs in other diseases remains unknown. Here, we review and summarize the essential characteristics and current findings concerning α7nAChRs in four common brain diseases (AD, Parkinson's disease, schizophrenia, and depression), which may elucidate the role of α7nAChRs and inform innovative research and novel treatments that target α7nAChRs in brain disease.

Neuroprotective effect of Ziziphi Spinosae Semen on rats with p-chlorophenylalanine-induced insomnia via activation of GABAA receptor.

Ziziphus jujuba var. spinosa (Bunge) Hu ex H.F.Chow [Rhamnaceae; Ziziphi Spinosae Semen (ZSS)] has attracted extensive attention as the first choice of traditional Chinese medicine in the treatment of insomnia. However, recent studies on the sleep-improving mechanism of ZSS have mainly focused on the role of single components. Thus, to further reveal the potential mechanism of ZSS, an assessment of its multiple constituents is necessary. In this study, ZSS extract (ZSSE) was obtained from ZSS via detailed modern extraction, separation, and purification technologies. The chemical constituents of ZSSE were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS). For in vivo experiments, a rat model of insomnia induced by p-chlorophenylalanine (PCPA) was established to investigate the potential effect and corresponding mechanism of ZSSE on improving sleep. Hematoxylin-eosin staining (HE) results revealed that the drug group showed prominent advantages over the model group in improving sleep. Moreover, the brain levels of γ-aminobutyric acid (GABA), glutamic acid (Glu), 5-hydroxytryptamine (5-HT), and dopamine (DA) were monitored via enzyme-linked immunosorbent assay (ELISA) to further study the sleep-improving mechanism of ZSSE. We found that sleep was effectively improved via upregulation of GABA and 5-HT and downregulation of Glu and DA. In addition, molecular mechanisms of ZSSE in improving sleep were studied by immunohistochemical analysis. The results showed that sleep was improved by regulating the expression levels of GABA receptor subunit alpha-1 (GABAARα1) and GABA acid receptor subunit gamma-2 (GABAARγ2) receptors in the hypothalamus and hippocampus tissue sections. Therefore, this work not only identified the active ingredients of ZSSE but also revealed the potential pharmacological mechanism of ZSSE for improving sleep, which may greatly stimulate the prospective development and application of ZSSE.

The Nicotinic Receptor Polymorphism rs16969968 Is Associated with Airway Remodeling and Inflammatory Dysregulation in COPD Patients.

Genome-wide association studies unveiled the associations between the single nucleotide polymorphism rs16969968 of CHRNA5, encoding the nicotinic acetylcholine receptor alpha5 subunit (α5SNP), and nicotine addiction, cancer, and COPD independently. Here, we investigated α5SNP-induced epithelial remodeling and inflammatory response in human COPD airways. We included 26 α5SNP COPD patients and 18 wild-type α5 COPD patients in a multi-modal study. A comparative histologic analysis was performed on formalin-fixed paraffin-embedded lung tissues. Isolated airway epithelial cells from bronchial brushings were cultivated in the air-liquid interface. Broncho-alveolar fluids were collected to detect inflammatory mediators. Ciliogenesis was altered in α5SNP COPD bronchial and bronchiolar epithelia. Goblet cell hyperplasia was exacerbated in α5SNP small airways. The broncho-alveolar fluids of α5SNP COPD patients exhibited an increase in inflammatory mediators. The involvement of the rs16969968 polymorphism in airway epithelial remodeling and related inflammatory response in COPD prompts the development of innovative personalized diagnostic and therapeutic strategies.

Immobilization of modular peptides on graphene cocktail for differentiation of human mesenchymal stem cells to hepatic-like cells.

In this study, two novel biomimetic modular peptide motifs based on the alpha-2 subunit of type IV collagen (CO4A2) were designed and immobilized on a graphene platform to imitate integrin and heparan sulfate- (HS-) binding proteins. The in silico study was used to design 9-mer K[KGDRGD]AG and 10-mer KK[SGDRGD]AG for testing designed Integrin-Binding Peptide (dIBP) and HS-Binding Peptide (dHBP). The virtual docking technique was used to optimize the peptide motifs and their relevant receptors. Molecular dynamic (MD) simulation was used to evaluate the stability of peptide-receptor complexes. The effect of the platform on the differentiation of human mesenchymal stem cells (hMSCs) to hepatic-like cells (HLCs) was evaluated. After differentiation, some hepatic cells’ molecular markers such as albumin, AFP, CK-18, and CK-19 were successfully followed. Graphene-heparan sulfate binding peptide (G-HSBP) enhances the mature hepatic markers’ expression instead of control (p ≤ 0.05). The pathological study showed that the designed platform is safe, and no adverse effects were seen till 21 days after implantation.

The Target Selects the Toxin: Specific Amino Acids in Snake-Prey Nicotinic Acetylcholine Receptors That Are Selectively Bound by King Cobra Venoms.

Snake venom is an adaptive ecological trait that has evolved primarily as a form of prey subjugation. Thus, the selection pressure for toxin diversification is exerted by the prey’s physiological targets, with this pressure being particularly acute for specialist feeders, such as the King Cobra species, all of which are snake-prey specialists. However, while extensive research has been undertaken to elucidate key amino acids that guide toxin structure–activity relationships, reciprocal investigations into the specific sites guiding prey-lineage selective effects have been lacking. This has largely been due to the lack of assay systems amenable to systematic amino acid replacements of targeted proteins in the prey’s physiological pathways. To fill this knowledge gap, we used a recently described approach based upon mimotope peptides corresponding to the orthosteric site of nicotinic acetylcholine receptor alpha-1 subunits, a major binding site for snake venom neurotoxins that cause flaccid paralysis. We investigated the venoms of four different types of King Cobra (Cambodian, Javan, Malaysian, and Thai). This approach allowed for the determination of the key amino acid positions in King Cobra snake prey that are selectively bound by the toxins, whereby replacing these amino acids in the snake-prey orthosteric site with those from lizards or rats resulted in a significantly lower level of binding by the venoms, while conversely replacing the lizard or rat amino acids with those from the snake at that position increased the binding. By doing such, we identified three negatively charged amino acids in the snake orthosteric site that are strongly bound by the positively charged neurotoxic three-finger toxins found in King Cobra venom. This study, thus, sheds light on the selection pressures exerted by a specialist prey item for the evolution of lineage-selective toxins.

Whole Transcriptome Profiling of the Effects of Cadmium on the Liver of the Xiangxi Yellow Heifer.

Cadmium (Cd) is a major heavy metal toxicant found in industrial zones. Humans and animals are exposed to it through their diet, which results in various physiological problems. In the current study, the toxic effects of Cd on the liver were investigated by whole-transcriptome sequencing (RNA-seq) of the livers of Xiangxi heifers fed a diet with excess Cd. We randomly divided six healthy heifers into two groups. The first group received a control diet, whereas the second group received Cd-exceeding diets for 100 days. After 100 days, the livers were collected. A total of 551 differentially expressed mRNAs, 24 differentially expressed miRNAs, and 169 differentially expressed lncRNAs were identified (p < 0.05, |log2FC| >1). Differentially expressed genes (DEGs) were analyzed by gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. We found that under Cd exposure, DEGs were enriched in the adenosine 5'-monophosphate–activated protein kinase pathway, which is involved in autophagy regulation, and the peroxisome proliferator–activated receptor pathway, which is involved in lipid metabolism. In addition, the apolipoprotein A4 gene, which has anti-inflammatory and antioxidant effects, the anti-apoptotic gene ATPase H+/K+ transporting the nongastric alpha2 subunit, and the cholesterol metabolism–associated gene endothelial lipase gene were significantly downregulated. C–X–C motif chemokine ligand 3, cholesterol 7α-hydroxylase, and stearoyl-CoA desaturase, which are involved in the development of fatty liver, were significantly upregulated. These genes revealed the main effects of Cd on the liver of Xiangxi yellow heifers. The current study provides insightful information regarding the DEGs involved in autophagy regulation, apoptosis, lipid metabolism, anti-inflammation, and antioxidant enzyme activity. These may serve as useful biomarkers for predicting and treating Cd-related diseases in the future.

Molecular characterization of alpha subunit 1 of sodium pump (ATP1A1) gene in Camelus dromedarius: its differential tissue expression potentially interprets the role in osmoregulation.

Dromedary or one-humped camel (Camelus dromedarius) is distinctively acclimatized to survive the arid conditions of the desert environment. It has an excellent ability to compete dehydration with substantial tolerance for rapid dehydration. Therefore, it offers an excellent model for studying osmoregulation. Molecular characterization of Na+/K+ ATPase as a central regulator of electrolyte normohemostasis affords a better understanding of this mechanism in camel. Here is the first to resolve the full-length of alpha-1 subunit of sodium pump (ATP1A1) gene with its differential expression in dromedary tissues. The nucleotide sequence for the recovered full cDNA of ATP1A1was submitted to the GenBank (NCBI GenBank accession #MW628635) and bioinformatically analyzed. The cDNA sequence was of 3760bp length with an open reading frame (ORF) of 3066bp encoding a putative 1021 amino acids polypeptide with a molecular mass of 112696Da. Blast search analysis revealed the shared high similarity of dromedary ATP1A1gene with other known ATP1A1genes in different species. The comparative analysis of its protein sequence confirmed the high identity with other mammalian ATP1A1 proteins. Further transcriptomic investigation for different organs was performed by real-time PCR to compare its level of expression among different organs. The results confirm a direct function between the ATP1A1 gene expression and the order of vital performance of these organs. The expression ofATP1A1mRNA in the adrenal gland and brain was significantly higher than that in the other organs. The noticed down expression in camel kidney concomitant with overexpression in the adrenal cortex might interpret how dromedary expels access sodium without water loss with relative high ability to restrain mineralocorticoid-induced sodium retention on drinking salty water. The results reflect the importance of sodium pump in these organs. Na+/K+ ATPase in the adrenal gland and brain than other organs.

374 Multi-omic Analysis Identifies a SPAK Kinase-regulated Ensemble of Choroid Plexus Ion Transport Proteins Relevant for Post-infectious Hydrocephalus

INTRODUCTION: Postinfectious hydrocephalus (PIH) is the most common cause of acquired hydrocephalus worldwide. Treatment is typically through CSF diversion via ventriculoperitoneal shunting, which has high rates of morbidity and is often unavailable in developing countries. METHODS: SPAK knockout (KO) rats were generated using Crispr/CAS9. Control and SPAK KO male rats were implanted with an infusion catheter to introduce E. coli (genetically engineered +/-LPS) or LPS into the lateral ventricles over 72 hours. CSF secretion was measured by catheter placement into the lateral ventricle, and ventricular size by MRI. ChP were harvested for immunohistochemistry (IHC), western blot (WB), genomic/proteomic analysis, and single-cell RNA sequencing (scRNAseq). To investigate SPAK-associated ChP targets, we purified the SPAK-protein complex, identified physical interactors using liquid chromatography/dual mass spectroscopy (LC-MS/MS), and validated top candidates by WB/IHC. RESULTS: Robust bumetanide-sensitive, SPAK-dependent, CSF hypersecretion and ventriculomegaly was observed and dependent on the presence of LPS. LPS triggered significant up-regulation of activating SPAK phosphorylation (Thr233/185), and the bumetanide-sensitive cation-chloride cotransporter NKCC1 (Thr203/207/212). LC-MS/MS pull-down assay additionally revealed three other important SPAK-bound ion transporters, alpha-1 subunit of Na/K ATPase, K+ channel KCNJ13/Kir7.1, and Cl- channel CLIC6. All SPAK-bound targets co-localized with pSPAK at the ChP apical membrane in LPS conditions. This interaction, as well as CSF hypersecretion and ventriculomegaly, were abrogated in LPS-treated SPAK KO animals. CONCLUSION: Multi-omic analysis identified a SPAK-regulated ensemble of ion transport proteins relevant for PIH at the ChP apical membrane. Given the recent development of SPAK kinase inhibitors, our findings uncover a targetable mechanism for the pharmacological treatment of PIH and potentially other forms of inflammation-associated acquired hydrocephalus.

Genetics of Primary Aldosteronism.

Primary aldosteronism is considered the commonest cause of secondary hypertension. In affected individuals, aldosterone is produced in an at least partially autonomous fashion in adrenal lesions (adenomas, [micro]nodules or diffuse hyperplasia). Over the past decade, next-generation sequencing studies have led to the insight that primary aldosteronism is largely a genetic disorder. Sporadic cases are due to somatic mutations, mostly in ion channels and pumps, and rare cases of familial hyperaldosteronism are caused by germline mutations in an overlapping set of genes. More than 90% of aldosterone-producing adenomas carry somatic mutations in K+ channel Kir3.4 (KCNJ5), Ca2+ channel CaV1.3 (CACNA1D), alpha-1 subunit of the Na+/K+ ATPase (ATP1A1), plasma membrane Ca2+ transporting ATPase 3 (ATP2B3), Ca2+ channel CaV3.2 (CACNA1H), Cl− channel ClC-2 (CLCN2), β-catenin (CTNNB1), and/or G-protein subunits alpha q/11 (GNAQ/11). Mutations in some of these genes have also been identified in aldosterone-producing (micro)nodules, suggesting a disease continuum from a single cell, acquiring a somatic mutation, via a nodule to adenoma formation, and from a healthy state to subclinical to overt primary aldosteronism. Individual glands can have multiple such lesions, and they can occur on both glands in bilateral disease. Familial hyperaldosteronism, typically with early onset, is caused by germline mutations in steroid 11-beta hydroxylase/ aldosterone synthase (CYP11B1/2), CLCN2, KCNJ5, CACNA1H, and CACNA1D.

Effects of palmitic acid on localization of embryo cell fate and blastocyst formation gene products.

As obese and overweight patients commonly display hyperlipidemia and are increasingly accessing fertility clinics for their conception needs, our studies are directed at understanding the effects of hyperlipidemia on early pregnancy. We have focused on investigating palmitic acid (PA) and oleic acid (OA) treatment alone and in combination from the mouse two-cell stage embryos as a model for understanding their effects on the mammalian preimplantation embryo. We recently reported that PA exerts a negative effect on mouse two-cell progression to the blastocyst stage, whereas OA co-treatment reverses that negative effect. In the present study, we hypothesized that PA treatment of mouse embryos would disrupt proper localization of cell fate determining and blastocyst formation gene products and that co-treatment with OA would reverse these effects. Our results demonstrate that PA treatment significantly (P < 0.05) reduces blastocyst development and cell number but did not prevent nuclear localization of YAP in outer cells. PA treatment significantly reduced the number of OCT4+ and CDX2+ nuclei. PA-treated embryos had lower expression of blastocyst formation proteins (E-cadherin, ZO-1 and Na/K-ATPase alpha1 subunit). Importantly, co-treatment of embryos with OA reversed PA-induced effects on blastocyst development and increased inner cell mass (ICM) and trophectoderm (TE) cell numbers and expression of blastocyst formation proteins. Our findings demonstrate that PA treatment does not impede cell fate gene localization but does disrupt proper blastocyst formation gene localization during mouse preimplantation development. OA treatment is protective and reverses PA’s detrimental effects. The results advance our understanding of the impact of FFA exposure on mammalian preimplantation development.

Subunit-specific autoantibodies in autoimmune autonomic ganglionopathy

Autoimmune Autonomic Ganglionopathy (AAG) is a disorder that causes autonomic failure and is associated with alpha3-ganglionic acetylcholine-receptor (gnACHR) antibodies. Assays that detect antibodies to whole gnACHR or subunits are available. We compared in-house subunit-specific immunoassays using bacterially-expressed alpha3 and beta4 subunits against an immunomodulation assay to detect antibodies in patients with AAG or control groups in a novel 2-step clinical-characteristic unblinding protocol. Only 1/8 patients with seropositive-AAG had subunit-specific antibodies, with sensitivity, specificity, false-negative and positive rates of 12.5, 85.2, 78.6 and 13.4% respectively. Subunit-specific antibody-derived false-positive results can lead to misdiagnosis, as autonomic failure is not specific to AAG.

Abstract 13166: Paraoxonase-3 Regulation of Cardiotonic Steroids Mediates Renal Injury and Dysfunction in a Dahl Salt Sensitive Model of Chronic Kidney Disease

Background: Paraoxonases (Pons) belong to a family of hydrolytic enzymes with 3 distinct isoforms: Pon 1, 2, and 3. While the physiologic substrates for Pons are unclear, their lactonase activity is considered central to their native physiological role. We have previously demonstrated an association between diminished Pon lactonase activity and worse clinical outcomes in volume expanded states such as chronic kidney disease (CKD). Furthermore, we have identified a novel reno-protective role for Pon3 in hypertensive renal disease. However, the underlying mechanism(s) are unknown. Cardiotonic steroids (CTS) are natriuretic hormones that are increased in CKD and promote renal inflammation and fibrosis via binding of their 17β-lactone ring to the alpha-1 subunit of the Na + /K + -ATPase (NKA-α-1). Therefore, we tested the hypothesis that reduction of Pon3 lactonase activity would lead to increased renal injury and dysfunction in a CTS induced rat model of CKD. Methods/Results: Experiments were performed on Dahl salt-sensitive rats (SS rats) and Pon3 knock-out rats on the Dahl salt-sensitive background (SS- Pon3 KO). First, CTS levels in the urine of SS control and SS- Pon3 KO rats were quantified using mass spectrometry. We found that SS- Pon3 KO rats had 59 times greater increase in 24-hour urine CTS levels vs SS rats. Next, to examine the role of Pon3 in mediating CTS induced renal injury, ten-week-old, age-matched male SS control and SS- Pon3 KO rats were administered the CTS telocinobufagin (TCB, 100 μg/kg/day s.q) for four weeks followed by assessment of blood pressure (tail cuff plethysmography) and renal function. We found that SS- Pon3 KO rats following TCB administration had significantly increased 24-hour urinary protein excretion (UPE) (50.73 ± 3.59vs 25.29 ± 2.07, p&lt;0.0001), as well as decreased FITC-Sinistrin monitored glomerular filtration rate (GFR) (0.80 ± 0.10 vs 1.16 ± 0.05, p&lt;0.01) compared to SS control rats, despite similar degrees of hypertension. Conclusion: These findings suggest that Pon3 plays a critical role in regulating CTS mediated renal dysfunction in vivo.

A quantitative trait variant in Gabra2 underlies increased methamphetamine stimulant sensitivity.

Psychostimulant (methamphetamine, cocaine) use disorders have a genetic component that remains mostly unknown. We conducted genome-wide quantitative trait locus (QTL) analysis of methamphetamine stimulant sensitivity. To facilitate gene identification, we employed a Reduced Complexity Cross between closely related C57BL/6 mouse substrains and examined maximum speed and distance traveled over 30 min following methamphetamine (2 mg/kg, i.p.). For maximum methamphetamine-induced speed following the second and third administration, we identified a single genome-wide significant QTL on chromosome 11 that peaked near the Cyfip2 locus (LOD = 3.5, 4.2; peak = 21 cM [36 Mb]). For methamphetamine-induced distance traveled following the first and second administration, we identified a genome-wide significant QTL on chromosome 5 that peaked near a functional intronic indel in Gabra2 coding for the alpha-2 subunit of the GABA-A receptor (LOD = 3.6–5.2; peak = 34–35 cM [66–67 Mb]). Striatal cis-expression QTL mapping corroborated Gabra2 as a functional candidate gene underlying methamphetamine-induced distance traveled. CRISPR/Cas9-mediated correction of the mutant intronic deletion on the C57BL/6J background to the wild-type C57BL/6NJ allele was sufficient to reduce methamphetamine-induced locomotor activity toward the wild-type C57BL/6NJ-like level, thus validating the quantitative trait variant (QTV). These studies show the power and efficiency of Reduced Complexity Crosses in identifying causal variants underlying complex traits. Functionally restoring Gabra2 expression decreased methamphetamine stimulant sensitivity and supports preclinical and human genetic studies implicating the GABA-A receptor in psychostimulant addiction-relevant traits. Importantly, our findings have major implications for studying psychostimulants in the C57BL/6J strain—the gold standard strain in biomedical research.