Bisphosphonate inhibitors of squalene synthase protect cells against cholesterol‐dependent cytolysins

Certain species of pathogenic bacteria damage tissues by secreting cholesterol‐dependent cytolysins, which form pores in the plasma membranes of animal cells. However, reducing cholesterol protects cells against these cytolysins. As the first committed step of cholesterol biosynthesis is catalyzed by squalene synthase, we explored whether inhibiting this enzyme protected cells against cholesterol‐dependent cytolysins. We first synthesized 22 different nitrogen‐containing bisphosphonate molecules that were designed to inhibit squalene synthase. Squalene synthase inhibition was quantified using a cell‐free enzyme assay, and validated by computer modeling of bisphosphonate molecules binding to squalene synthase. The bisphosphonates were then screened for their ability to protect HeLa cells against the damage caused by the cholesterol‐dependent cytolysin, pyolysin. The most effective bisphosphonate reduced pyolysin‐induced leakage of lactate dehydrogenase into cell supernatants by >80%, and reduced pyolysin‐induced cytolysis from >75% to <25%. In addition, this bisphosphonate reduced pyolysin‐induced leakage of potassium from cells, limited changes in the cytoskeleton, prevented mitogen‐activated protein kinases cell stress responses, and reduced cellular cholesterol. The bisphosphonate also protected cells against another cholesterol‐dependent cytolysin, streptolysin O, and protected lung epithelial cells and primary dermal fibroblasts against cytolysis. Our findings imply that treatment with bisphosphonates that inhibit squalene synthase might help protect tissues against pathogenic bacteria that secrete cholesterol‐dependent cytolysins

Structure-Related Differences between Cytochrome Oxidase I Proteins in a Stable Heteroplasmic Mitochondrial System

open5siMIUR, programma SIR, progetto n. RBSI14G0P5 "MItochoNdria, Germ Line and sEx" (MINGLE)Many bivalve species have two types of mitochondrial DNA passed independently through the female line (F genome) and male line (M genome). Here we study the cytochrome oxidase I protein in such bivalve species and provide evidence for differences between the F and M proteins in amino acid property values, particularly relating to hydrophobicity and helicity. The magnitude of these differences varies between different regions of the protein and the change from the ancestor is most marked in the M protein. The observed changes occur in parallel and in the same direction in the different species studied. Two possible causes are considered, first relaxation of purifying selection with drift and second positive selection. These may operate in different ways in different regions of the protein. Many different amino acid substitutions contribute in a small way to the observed variation, but substitutions involving alanine and serine have a quantitatively large effect. Some of these substitutions are potential targets for phosphorylation and some are close to residues of functional importance in the catalytic mechanism. We propose that the observed changes in the F and M proteins might contribute to functional differences between them relating to ATP production and mitochondrial membrane potential with implications for sperm function.openSkibinski, David O F; Ghiselli, Fabrizio; Diz, Angel P; Milani, Liliana; Mullins, Jonathan G LSkibinski, David O F; Ghiselli, Fabrizio; Diz, Angel P; Milani, Liliana; Mullins, Jonathan G

Alternating hemiplegia of childhood-related neural and behavioural phenotypes in Na+,K+-ATPase α3 missense mutant mice

Missense mutations in ATP1A3 encoding Na(+),K(+)-ATPase α3 have been identified as the primary cause of alternating hemiplegia of childhood (AHC), a motor disorder with onset typically before the age of 6 months. Affected children tend to be of short stature and can also have epilepsy, ataxia and learning disability. The Na(+),K(+)-ATPase has a well-known role in maintaining electrochemical gradients across cell membranes, but our understanding of how the mutations cause AHC is limited. Myshkin mutant mice carry an amino acid change (I810N) that affects the same position in Na(+),K(+)-ATPase α3 as I810S found in AHC. Using molecular modelling, we show that the Myshkin and AHC mutations display similarly severe structural impacts on Na(+),K(+)-ATPase α3, including upon the K(+) pore and predicted K(+) binding sites. Behavioural analysis of Myshkin mice revealed phenotypic abnormalities similar to symptoms of AHC, including motor dysfunction and cognitive impairment. 2-DG imaging of Myshkin mice identified compromised thalamocortical functioning that includes a deficit in frontal cortex functioning (hypofrontality), directly mirroring that reported in AHC, along with reduced thalamocortical functional connectivity. Our results thus provide validation for missense mutations in Na(+),K(+)-ATPase α3 as a cause of AHC, and highlight Myshkin mice as a starting point for the exploration of disease mechanisms and novel treatments in AHC

Bile acid biosynthesis in Smith-Lemli-Opitz syndrome bypassing cholesterol: Potential importance of pathway intermediates

Bile acids are the end products of cholesterol metabolism secreted into bile. They are essential for the absorption of lipids and lipid soluble compounds from the intestine. Here we have identified a series of unusual Δ5-unsaturated bile acids in plasma and urine of patients with Smith-Lemli-Opitz syndrome (SLOS), a defect in cholesterol biosynthesis resulting in elevated levels of 7-dehydrocholesterol (7-DHC), an immediate precursor of cholesterol. Using liquid chromatography – mass spectrometry (LC-MS) we have uncovered a pathway of bile acid biosynthesis in SLOS avoiding cholesterol starting with 7-DHC and proceeding through 7-oxo and 7β-hydroxy intermediates. This pathway also occurs to a minor extent in healthy humans, but elevated levels of pathway intermediates could be responsible for some of the features SLOS. The pathway is also active in SLOS affected pregnancies as revealed by analysis of amniotic fluid. Importantly, intermediates in the pathway, 25-hydroxy-7-oxocholesterol, (25R)26-hydroxy-7-oxocholesterol, 3β-hydroxy-7-oxocholest-5-en-(25R)26-oic acid and the analogous 7β-hydroxysterols are modulators of the activity of Smoothened (Smo), an oncoprotein that mediates Hedgehog (Hh) signalling across membranes during embryogenesis and in the regeneration of postembryonic tissue. Computational docking of the 7-oxo and 7β-hydroxy compounds to the extracellular cysteine rich domain of Smo reveals that they bind in the same groove as both 20S-hydroxycholesterol and cholesterol, known activators of the Hh pathway

The glycinergic system in human startle disease: a genetic screening approach.

Human startle disease, also known as hyperekplexia (OMIM 149400), is a paroxysmal neurological disorder caused by defects in glycinergic neurotransmission. Hyperekplexia is characterised by an exaggerated startle reflex in response to tactile or acoustic stimuli which first presents as neonatal hypertonia, followed in some with episodes of life-threatening infantile apnoea. Genetic screening studies have demonstrated that hyperekplexia is genetically heterogeneous with several missense and nonsense mutations in the postsynaptic glycine receptor (GlyR) alpha1 subunit gene (GLRA1) as the primary cause. More recently, missense, nonsense and frameshift mutations have also been identified in the glycine transporter GlyT2 gene, SLC6A5, demonstrating a presynaptic component to this disease. Further mutations, albeit rare, have been identified in the genes encoding the GlyR beta subunit (GLRB), collybistin (ARHGEF9) and gephyrin (GPHN) - all of which are postsynaptic proteins involved in orchestrating glycinergic neurotransmission. In this review, we describe the clinical ascertainment aspects, phenotypic considerations and the downstream molecular genetic tools utilised to analyse both presynaptic and postsynaptic components of this heterogeneous human neurological disorder. Moreover, we will describe how the ancient startle response is the preserve of glycinergic neurotransmission and how animal models and human hyperekplexia patients have provided synergistic evidence that implicates this inhibitory system in the control of startle reflexes

Choriocapillaris and Choroidal Microvasculature Imaging with Ultrahigh Speed OCT Angiography

We demonstrate in vivo choriocapillaris and choroidal microvasculature imaging in normal human subjects using optical coherence tomography (OCT). An ultrahigh speed swept source OCT prototype at 1060 nm wavelengths with a 400 kHz A-scan rate is developed for three-dimensional ultrahigh speed imaging of the posterior eye. OCT angiography is used to image three-dimensional vascular structure without the need for exogenous fluorophores by detecting erythrocyte motion contrast between OCT intensity cross-sectional images acquired rapidly and repeatedly from the same location on the retina. En face OCT angiograms of the choriocapillaris and choroidal vasculature are visualized by acquiring cross-sectional OCT angiograms volumetrically via raster scanning and segmenting the three-dimensional angiographic data at multiple depths below the retinal pigment epithelium (RPE). Fine microvasculature of the choriocapillaris, as well as tightly packed networks of feeding arterioles and draining venules, can be visualized at different en face depths. Panoramic ultra-wide field stitched OCT angiograms of the choriocapillaris spanning ~32 mm on the retina show distinct vascular structures at different fundus locations. Isolated smaller fields at the central fovea and ~6 mm nasal to the fovea at the depths of the choriocapillaris and Sattler's layer show vasculature structures consistent with established architectural morphology from histological and electron micrograph corrosion casting studies. Choriocapillaris imaging was performed in eight healthy volunteers with OCT angiograms successfully acquired from all subjects. These results demonstrate the feasibility of ultrahigh speed OCT for in vivo dye-free choriocapillaris and choroidal vasculature imaging, in addition to conventional structural imaging.National Institutes of Health (U.S.) (NIH R01-EY011289-27)National Institutes of Health (U.S.) (NIH R01-EY013178-12)National Institutes of Health (U.S.) (NIH R44-EY022864-01)National Institutes of Health (U.S.) (NIH R01-CA075289-16)United States. Air Force Office of Scientific Research (AFOSR FA9550-10-1-0551)United States. Air Force Office of Scientific Research (AFOSR FA9550-12-1-0499

In Vitro and In Silico Analyses of the Inhibition of Human Aldehyde Oxidase by Bazedoxifene, Lasofoxifene, and Structural Analogues

Aldehyde oxidase (AOX1) is a molybdo-flavoprotein and has emerged as a drug-metabolizing enzyme of potential therapeutic importance because drugs have been identified as AOX1 substrates. Selective oestrogen receptor modulators (SERM), which are drugs used to treat and prevent various conditions, differentially inhibit AOX1 catalytic activity. Tamoxifen, raloxifene, and nafoxidine are selective oestrogen receptor modulators (SERMs) reported to inhibit the catalytic activity of human aldehyde oxidase 1 (AOX1). How these drugs interact with AOX1 and whether other SERMs inhibit this drug-metabolizing enzyme are not known. Therefore, a detailed in vitro and in silico study involving parent drugs and their analogues was conducted to investigate the effect of specific SERMs, particularly acolbifene, bazedoxifene, and lasofoxifene on AOX1 catalytic activity, as assessed by carbazeran 4-oxidation, an AOX1-selective catalytic marker. The rank-order in the potency (based on IC50 values) of AOX1 inhibition by SERMs was raloxifene > bazedoxifene ~ lasofoxifene > tamoxifen > acolbifene. Inhibition of liver cytosolic AOX1 by bazedoxifene, lasofoxifene, and tamoxifen was competitive, whereas that by raloxifene was noncompetitive. Loss of 1-azepanylethyl group increased the inhibitory potency of bazedoxifene, whereas the N-oxide group decreased it. The 7-hydroxy group and the substituted pyrrolidine ring attached to the tetrahydronaphthalene structure contributed to AOX1 inhibition by lasofoxifene. These results are supported by molecular docking simulations in terms of predicted binding modes, encompassing binding orientation and efficiency, and analysis of key interactions, particularly hydrogen bonds. The extent of AOX1 inhibition by bazedoxifene was increased by estrone sulfate and estrone. In summary, SERMs differentially inhibited human AOX1 catalytic activity. Structural features of bazedoxifene and lasofoxifene contributed to AOX1 inhibition, whereas those of acolbifene rendered it considerably less susceptible to AOX1 inhibition. Overall, our novel biochemical findings and molecular docking analyses provide new insights into the interaction between SERMs and AOX1. Structural features of bazedoxifene and lasofoxifene contribute to AOX1 inhibition, whereas those of acolbifene render it considerably less susceptible to AOX1 inhibition. Our novel biochemical findings, together with molecular docking analyses, provide new insights into the differential inhibitory effect of SERMs on the catalytic activity of human AOX1, how SERMs bind to AOX1, and increase our understanding of the AOX1 pharmacophore in the inhibition of AOX1 by drugs and other chemicals

Association of Genetic Variants With Primary Open-Angle Glaucoma Among Individuals With African Ancestry.

Importance:Primary open-angle glaucoma presents with increased prevalence and a higher degree of clinical severity in populations of African ancestry compared with European or Asian ancestry. Despite this, individuals of African ancestry remain understudied in genomic research for blinding disorders. Objectives:To perform a genome-wide association study (GWAS) of African ancestry populations and evaluate potential mechanisms of pathogenesis for loci associated with primary open-angle glaucoma. Design, Settings, and Participants:A 2-stage GWAS with a discovery data set of 2320 individuals with primary open-angle glaucoma and 2121 control individuals without primary open-angle glaucoma. The validation stage included an additional 6937 affected individuals and 14 917 unaffected individuals using multicenter clinic- and population-based participant recruitment approaches. Study participants were recruited from Ghana, Nigeria, South Africa, the United States, Tanzania, Britain, Cameroon, Saudi Arabia, Brazil, the Democratic Republic of the Congo, Morocco, Peru, and Mali from 2003 to 2018. Individuals with primary open-angle glaucoma had open iridocorneal angles and displayed glaucomatous optic neuropathy with visual field defects. Elevated intraocular pressure was not included in the case definition. Control individuals had no elevated intraocular pressure and no signs of glaucoma. Exposures:Genetic variants associated with primary open-angle glaucoma. Main Outcomes and Measures:Presence of primary open-angle glaucoma. Genome-wide significance was defined as P &lt; 5 × 10-8 in the discovery stage and in the meta-analysis of combined discovery and validation data. Results:A total of 2320 individuals with primary open-angle glaucoma (mean [interquartile range] age, 64.6 [56-74] years; 1055 [45.5%] women) and 2121 individuals without primary open-angle glaucoma (mean [interquartile range] age, 63.4 [55-71] years; 1025 [48.3%] women) were included in the discovery GWAS. The GWAS discovery meta-analysis demonstrated association of variants at amyloid-β A4 precursor protein-binding family B member 2 (APBB2; chromosome 4, rs59892895T&gt;C) with primary open-angle glaucoma (odds ratio [OR], 1.32 [95% CI, 1.20-1.46]; P = 2 × 10-8). The association was validated in an analysis of an additional 6937 affected individuals and 14 917 unaffected individuals (OR, 1.15 [95% CI, 1.09-1.21]; P &lt; .001). Each copy of the rs59892895*C risk allele was associated with increased risk of primary open-angle glaucoma when all data were included in a meta-analysis (OR, 1.19 [95% CI, 1.14-1.25]; P = 4 × 10-13). The rs59892895*C risk allele was present at appreciable frequency only in African ancestry populations. In contrast, the rs59892895*C risk allele had a frequency of less than 0.1% in individuals of European or Asian ancestry. Conclusions and Relevance:In this genome-wide association study, variants at the APBB2 locus demonstrated differential association with primary open-angle glaucoma by ancestry. If validated in additional populations this finding may have implications for risk assessment and therapeutic strategies

De novo mutations in GRIN1 cause extensive bilateral polymicrogyria

Polymicrogyria is a malformation of cortical development. The aetiology of polymicrogyria remains poorly understood. Using whole-exome sequencing we found de novo heterozygous missense GRIN1 mutations in 2 of 57 parent-offspring trios with polymicrogyria. We found nine further de novo missense GRIN1 mutations in additional cortical malformation patients. Shared features in the patients were extensive bilateral polymicrogyria associated with severe developmental delay, postnatal microcephaly, cortical visual impairment and intractable epilepsy. GRIN1 encodes GluN1, the essential subunit of the N-methyl-d-aspartate receptor. The polymicrogyria-associated GRIN1 mutations tended to cluster in the S2 region (part of the ligand-binding domain of GluN1) or the adjacent M3 helix. These regions are rarely mutated in the normal population or in GRIN1 patients without polymicrogyria. Using two-electrode and whole-cell voltage-clamp analysis, we showed that the polymicrogyria-associated GRIN1 mutations significantly alter the in vitro activity of the receptor. Three of the mutations increased agonist potency while one reduced proton inhibition of the receptor. These results are striking because previous GRIN1 mutations have generally caused loss of function, and because N-methyl-d-aspartate receptor agonists have been used for many years to generate animal models of polymicrogyria. Overall, our results expand the phenotypic spectrum associated with GRIN1 mutations and highlight the important role of N-methyl-d-aspartate receptor signalling in the pathogenesis of polymicrogyria

Specific Inhibition of Phosphodiesterase-4B Results in Anxiolysis and Facilitates Memory Acquisition

Cognitive dysfunction is a core feature of dementia and a prominent feature in psychiatric disease. As non-redundant regulators of intracellular cAMP gradients, phosphodiesterases (PDE) mediate fundamental aspects of brain function relevant to learning, memory, and higher cognitive functions. Phosphodiesterase-4B (PDE4B) is an important phosphodiesterase in the hippocampal formation, is a major Disrupted in Schizophrenia 1 (DISC1) binding partner and is itself a risk gene for psychiatric illness. To define the effects of specific inhibition of the PDE4B subtype, we generated mice with a catalytic domain mutant form of PDE4B (Y358C) that has decreased ability to hydrolyze cAMP. Structural modelling predictions of decreased function and impaired binding with DISC1 were confirmed in cell assays. Phenotypic characterization of the PDE4BY358C mice revealed facilitated phosphorylation of CREB, decreased binding to DISC1, and upregulation of DISC1 and β-Arrestin in hippocampus and amygdala. In behavioural assays, PDE4BY358C mice displayed decreased anxiety and increased exploration, as well as cognitive enhancement across several tests of learning and memory, consistent with synaptic changes including enhanced long-term potentiation and impaired depotentiation ex vivo. PDE4BY358C mice also demonstrated enhanced neurogenesis. Contextual fear memory, though intact at 24 hours, was decreased at 7 days in PDE4BY358C mice, an effect replicated pharmacologically with a non-selective PDE4 inhibitor, implicating cAMP signalling by PDE4B in a very late phase of consolidation. No effect of the PDE4BY358C mutation was observed in the pre-pulse inhibition and forced swim tests. Our data establish specific inhibition of PDE4B as a promising therapeutic approach for disorders of cognition and anxiety, and a putative target for pathological fear memory