Murine cathepsin D deficiency is associated with dysmyelination/myelin disruption and accumulation of cholesteryl esters in the brain

Cathepsin D (CTSD) deficiencies are fatal neurological diseases that in human infants and in sheep are characterized by extreme loss of neurons and myelin. To date, similar morphological evidence for myelin disruption in CTSD knockout mice has not been reported. Here, we show that CTSD deficiency leads to pronounced myelin changes in the murine brain: myelin-related proteolipid protein and myelin basic protein were both markedly reduced at postnatal day 24, and the amount of lipids characteristically high in myelin (e.g. plasmalogen-derived alkenyl chains and glycosphingolipid-derived 20- and 24-carbon acyl chains) were significantly lowered compared with controls. These changes were accompanied by ultrastructural alterations of myelin, including significant thinning of myelin sheaths. Furthermore, in CTSD knockout brains there was a pronounced accumulation of cholesteryl esters and abnormal levels of proteins related to cholesterol transport, with an increased content of apolipoprotein E and a reduced content of ATP-binding cassette transporter A1. These results provide evidence for dysmyelination and altered trafficking of cholesterol in brains of CTSD knockout mice, and warrant further studies on the role of lipid metabolism in the pathogenesis of CTSD deficiencies.     

Pharmacologic inhibition of S-nitrosoglutathione reductase protects against experimental asthma in BALB/c mice through attenuation of both bronchoconstriction and inflammation

Background

S-nitrosoglutathione (GSNO) serves as a reservoir for nitric oxide (NO) and thus is a key homeostatic regulator of airway smooth muscle tone and inflammation. Decreased levels of GSNO in the lungs of asthmatics have been attributed to increased GSNO catabolism via GSNO reductase (GSNOR) leading to loss of GSNO- and NO- mediated bronchodilatory and anti-inflammatory actions. GSNOR inhibition with the novel small molecule, N6022, was explored as a therapeutic approach in an experimental model of asthma.

Methods

Female BALB/c mice were sensitized and subsequently challenged with ovalbumin (OVA). Efficacy was determined by measuring both airway hyper-responsiveness (AHR) upon methacholine (MCh) challenge using whole body plethysmography and pulmonary eosinophilia by quantifying the numbers of these cells in the bronchoalveolar lavage fluid (BALF). Several other potential biomarkers of GSNOR inhibition were measured including levels of nitrite, cyclic guanosine monophosphate (cGMP), and inflammatory cytokines, as well as DNA binding activity of nuclear factor kappa B (NFκB). The dose response, onset of action, and duration of action of a single intravenous dose of N6022 given from 30 min to 48 h prior to MCh challenge were determined and compared to effects in mice not sensitized to OVA. The direct effect of N6022 on airway smooth muscle tone also was assessed in isolated rat tracheal rings.

Results

N6022 attenuated AHR (ED₅₀ of 0.015?±?0.002 mg/kg; Mean?±?SEM) and eosinophilia. Effects were observed from 30 min to 48 h after treatment and were comparable to those achieved with three inhaled doses of ipratropium plus albuterol used as the positive control. N6022 increased BALF nitrite and plasma cGMP, while restoring BALF and plasma inflammatory markers toward baseline values. N6022 treatment also attenuated the OVA-induced increase in NFκB activation. In rat tracheal rings, N6022 decreased contractile responses to MCh.

Conclusions

The significant bronchodilatory and anti-inflammatory actions of N6022 in the airways are consistent with restoration of GSNO levels through GSNOR inhibition. GSNOR inhibition may offer a therapeutic approach for the treatment of asthma and other inflammatory lung diseases. N6022 is currently being evaluated in clinical trials for the treatment of inflammatory lung disease.     

ADH IB Expression,but Not ADH III,Is Decreased in Human Lung Cancer

Endogenous S-nitrosothiols, including S-nitrosoglutathione (GSNO), mediate nitric oxide (NO)-based signaling, inflammatory responses, and smooth muscle function. Reduced GSNO levels have been implicated in several respiratory diseases, and inhibition of GSNO reductase, (GSNOR) the primary enzyme that metabolizes GSNO, represents a novel approach to treating inflammatory lung diseases. Recently, an association between decreased GSNOR expression and human lung cancer risk was proposed in part based on immunohistochemical staining using a polyclonal GSNOR antibody. GSNOR is an isozyme of the alcohol dehydrogenase (ADH) family, and we demonstrate that the antibody used in those studies cross reacts substantially with other ADH proteins and may not be an appropriate reagent. We evaluated human lung cancer tissue arrays using monoclonal antibodies highly specific for human GSNOR with minimal cross reactivity to other ADH proteins. We verified the presence of GSNOR in ≥85% of specimens examined, and extensive analysis of these samples demonstrated no difference in GSNOR protein expression between cancerous and normal lung tissues. Additionally, GSNOR and other ADH mRNA levels were evaluated quantitatively in lung cancer cDNA arrays by qPCR. Consistent with our immunohistochemical findings, GSNOR mRNA levels were not changed in lung cancer tissues, however the expression levels of other ADH genes were decreased. ADH IB mRNA levels were reduced (>10-fold) in 65% of the lung cancer cDNA specimens. We conclude that the previously reported results showed an incorrect association of GSNOR and human lung cancer risk, and a decrease in ADH IB, rather than GSNOR, correlates with human lung cancer.     

Structure-activity relationship of pyrrole based S-nitrosoglutathione reductase inhibitors: carboxamide modification

The enzyme S-nitrosoglutathione reductase (GSNOR) is a member of the alcohol dehydrogenase family (ADH) that regulates the levels of S-nitrosothiols (SNOs) through catabolism of S-nitrosoglutathione (GSNO). GSNO and SNOs are implicated in the pathogenesis of many diseases including those in respiratory, gastrointestinal, and cardiovascular systems. The pyrrole based N6022 was recently identified as a potent, selective, reversible, and efficacious GSNOR inhibitor which is currently in clinical development for acute asthma. We describe here the synthesis and structure-activity relationships (SAR) of novel pyrrole based analogs of N6022 focusing on carboxamide modifications on the pendant N-phenyl moiety. We have identified potent and novel GSNOR inhibitors that demonstrate efficacy in an ovalbumin (OVA) induced asthma model in mice.     

MLN64 is involved in actin-mediated dynamics of late endocytic organelles

MLN64 is a late endosomal cholesterol-binding membrane protein of an unknown function. Here, we show that MLN64 depletion results in the dispersion of late endocytic organelles to the cell periphery similarly as upon pharmacological actin disruption. The dispersed organelles in MLN64 knockdown cells exhibited decreased association with actin and the Arp2/3 complex subunit p34-Arc. MLN64 depletion was accompanied by impaired fusion of late endocytic organelles and delayed cargo degradation. MLN64 overexpression increased the number of actin and p34-Arc-positive patches on late endosomes, enhanced the fusion of late endocytic organelles in an actin-dependent manner, and stimulated the deposition of sterol in late endosomes harboring the protein. Overexpression of wild-type MLN64 was capable of rescuing the endosome dispersion in MLN64-depleted cells, whereas mutants of MLN64 defective in cholesterol binding were not, suggesting a functional connection between MLN64-mediated sterol transfer and actin-dependent late endosome dynamics. We propose that local sterol enrichment by MLN64 in the late endosomal membranes facilitates their association with actin, thereby governing actin-dependent fusion and degradative activity of late endocytic organelles.     

Discovery of potent and novel S-nitrosoglutathione reductase inhibitors devoid of cytochrome P450 activities

The pyrrole based N6022 was recently identified as a potent, selective, reversible, and efficacious S-nitrosoglutathione reductase (GSNOR) inhibitor and is currently undergoing clinical development for the treatment of acute asthma. GSNOR is a member of the alcohol dehydrogenase family (ADH) and regulates the levels of S-nitrosothiols (SNOs) through catabolism of S-nitrosoglutathione (GSNO). Reduced levels of GSNO, as well as other nitrosothiols (SNOs), have been implicated in the pathogenesis of many diseases including those of the respiratory, cardiovascular, and gastrointestinal systems. Preservation of endogenous SNOs through GSNOR inhibition presents a novel therapeutic approach with broad applicability. We describe here the synthesis and structure-activity relationships (SAR) of novel pyrrole based analogues of N6022 focusing on removal of cytochrome P450 inhibition activities. We identified potent and novel GSNOR inhibitors having reduced CYP inhibition activities and demonstrated efficacy in a mouse ovalbumin (OVA) model of asthma.     

Auxin promotes susceptibility to Pseudomonas syringae via a mechanism independent of suppression of salicylic acid‐mediated defenses

Auxin is a key plant growth regulator that also impacts plant–pathogen interactions. Several lines of evidence suggest that the bacterial plant pathogen Pseudomonas syringae manipulates auxin physiology in Arabidopsis thaliana to promote pathogenesis. Pseudomonas syringae strategies to alter host auxin biology include synthesis of the auxin indole‐3‐acetic acid (IAA) and production of virulence factors that alter auxin responses in host cells. The application of exogenous auxin enhances disease caused by P. syringae strain DC3000. This is hypothesized to result from antagonism between auxin and salicylic acid (SA), a major regulator of plant defenses, but this hypothesis has not been tested in the context of infected plants. We further investigated the role of auxin during pathogenesis by examining the interaction of auxin and SA in the context of infection in plants with elevated endogenous levels of auxin. We demonstrated that elevated IAA biosynthesis in transgenic plants overexpressing the YUCCA 1 (YUC1) auxin biosynthesis gene led to enhanced susceptibility to DC3000. Elevated IAA levels did not interfere significantly with host defenses, as effector‐triggered immunity was active in YUC1‐overexpressing plants, and we observed only minor effects on SA levels and SA‐mediated responses. Furthermore, a plant line carrying both the YUC1‐overexpression transgene and the salicylic acid induction deficient 2 (sid2) mutation, which impairs SA synthesis, exhibited additive effects of enhanced susceptibility from both elevated auxin levels and impaired SA‐mediated defenses. Thus, in IAA overproducing plants, the promotion of pathogen growth occurs independently of suppression of SA‐mediated defenses.     

Allele exchange at the EPSPS locus confers glyphosate tolerance in cassava

Effective weed control can protect yields of cassava (Manihot esculenta) storage roots. Farmers could benefit from using herbicide with a tolerant cultivar. We applied traditional transgenesis and gene editing to generate robust glyphosate tolerance in cassava. By comparing promoters regulating expression of transformed 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) genes with various paired amino acid substitutions, we found that strong constitutive expression is required to achieve glyphosate tolerance during in vitro selection and in whole cassava plants. Using strategies that exploit homologous recombination (HR) and nonhomologous end‐joining (NHEJ) DNA repair pathways, we precisely introduced the best‐performing allele into the cassava genome, simultaneously creating a promoter swap and dual amino acid substitutions at the endogenous EPSPS locus. Primary EPSPS‐edited plants were phenotypically normal, tolerant to high doses of glyphosate, with some free of detectable T‐DNA integrations. Our methods demonstrate an editing strategy for creating glyphosate tolerance in crop plants and demonstrate the potential of gene editing for further improvement of cassava.