A genome-wide RNAi screen identifies potential drug targets in a C. elegans model of α1-antitrypsin deficiency.

α1-Antitrypsin deficiency (ATD) is a common genetic disorder that can lead to end-stage liver and lung disease. Although liver transplantation remains the only therapy currently available, manipulation of the proteostasis network (PN) by small molecule therapeutics offers great promise. To accelerate the drug-discovery process for this disease, we first developed a semi-automated high-throughput/content-genome-wide RNAi screen to identify PN modifiers affecting the accumulation of the α1-antitrypsin Z mutant (ATZ) in a Caenorhabditis elegans model of ATD. We identified 104 PN modifiers, and these genes were used in a computational strategy to identify human ortholog-ligand pairs. Based on rigorous selection criteria, we identified four FDA-approved drugs directed against four different PN targets that decreased the accumulation of ATZ in C. elegans. We also tested one of the compounds in a mammalian cell line with similar results. This methodology also proved useful in confirming drug targets in vivo, and predicting the success of combination therapy. We propose that small animal models of genetic disorders combined with genome-wide RNAi screening and computational methods can be used to rapidly, economically and strategically prime the preclinical discovery pipeline for rare and neglected diseases with limited therapeutic options.

A C. elegans model of human α1-antitrypsin deficiency links components of the RNAi pathway to misfolded protein turnover.

The accumulation of serpin oligomers and polymers within the endoplasmic reticulum (ER) causes cellular injury in patients with the classical form α1-antitrypsin deficiency (ATD). To better understand the cellular and molecular genetic aspects of this disorder, we generated transgenic C. elegans strains expressing either the wild-type (ATM) or Z mutant form (ATZ) of the human serpin fused to GFP. Animals secreted ATM, but retained polymerized ATZ within dilated ER cisternae. These latter animals also showed slow growth, smaller brood sizes and decreased longevity; phenotypes observed in ATD patients or transgenic mouse lines expressing ATZ. Similar to mammalian models, ATZ was disposed of by autophagy and ER-associated degradation pathways. Mutant strains defective in insulin signaling (daf-2) also showed a marked decrease in ATZ accumulation. Enhanced ATZ turnover was associated with the activity of two proteins central to systemic/exogenous (exo)-RNAi pathway: the dsRNA importer, SID-1 and the argonaute, RDE-1. Animals with enhanced exo-RNAi activity (rrf-3 mutant) phenocopied the insulin signaling mutants and also showed increased ATZ turnover. Taken together, these studies allude to the existence of a novel proteostasis pathway that mechanistically links misfolded protein turnover to components of the systemic RNAi machinery.

A high-content assay for identifying small molecules that reprogram C. elegans germ cell fate.

Recent breakthrough discoveries have shown that committed cell fates can be reprogrammed by genetic, chemical and environmental manipulations. The germline of the nematode Caenorhabditis elegans provides a tractable system for studying cell fate reprogramming within the context of a whole organism. To explore the possibility of using C. elegans in high-throughput screens (HTS), we developed a high-throughput workflow for testing compounds that modulate cell fate reprogramming. We utilized puf-8; lip-1 mutants that have enhanced MPK-1 (an ERK homolog)/MAP kinase (MAPK) signaling. Wild-type C. elegans hermaphrodites produce both sperm and oocytes, and are thus self-fertile. However, puf-8; lip-1 mutants produce only sperm and are sterile. Notably, compounds that pharmacologically down-regulate MPK-1 (an ERK homolog)/MAP kinase (MAPK) signaling are able to reprogram germ cell fate and restore fertility to these animals. puf-8; lip-1 mutants provide numerous challenges for HTS. First, they are sterile as homozygotes and must be maintained as heterozygotes using a balancer chromosome. Second, homozygous animals for experimentation must be physically separated from the rest of the population. Third, a high quality, high-content assay has not been developed to measure compound effects on germ cell fate reprogramming. Here we describe a semi-automated high-throughput workflow that enables effective sorting of homozygous puf-8; lip-1 mutants into 384-well plates using the COPAS™ BIOSORT. In addition, we have developed an image-based assay for rapidly measuring germ cell reprogramming by measuring the number of viable progeny in wells. The methods presented in this report enable the use of puf-8; lip-1 mutants in HTS campaigns for chemical modulators of germ cell reprogramming within the context of a whole organism.

Profile of sleep disturbances in patients with recurrent depressive disorder or bipolar affective disorder in a tertiary sleep disorders service.

Bidirectional relationship between sleep disturbances and affective disorders is increasingly recognised, but its underlying mechanisms are far from clear, and there is a scarcity of studies that report on sleep disturbances in recurrent depressive disorder (RDD) and bipolar affective disorder (BPAD). To address this, we conducted a retrospective study of polysomnographic and clinical records of patients presenting to a tertiary sleep disorders clinic with affective disorders. Sixty-three BPAD patients (32 female; mean age ± S.D.: 41.8 ± 12.4 years) and 126 age- and gender-matched RDD patients (62 female; 41.5 ± 12.8) were studied. Whilst no significant differences were observed in sleep macrostructure parameters between BPAD and RDD patients, major differences were observed in comorbid sleep and physical disorders, both of which were higher in BPAD patients. Two most prevalent sleep disorders, namely obstructive sleep apnoea (OSA) (BPAD 50.8.0% vs RDD 29.3%, P = 0.006) and insomnia (BPAD 34.9% vs RDD 15.0%, P = 0.005) were found to be strongly linked with BPAD. In summary, in our tertiary sleep clinic cohort, no overt differences in the sleep macrostructure between BPAD and RDD patients were demonstrated. However, OSA and insomnia, two most prevalent sleep disorders, were found significantly more prevalent in patients with BPAD, by comparison to RDD patients. Also, BPAD patients presented with significantly more severe OSA, and with higher overall physical co-morbidity. Thus, our findings suggest an unmet/hidden need for earlier diagnosis of those with BPAD.

Endothelial cyclooxygenase-1 paradoxically drives local vasoconstriction and atherogenesis despite underpinning prostacyclin generation.

Endothelial cyclooxygenase-1-derived prostanoids, including prostacyclin, have clear cardioprotective roles associated with their anti-thrombotic potential but have also been suggested to have paradoxical pathological activities within arteries. To date it has not been possible to test the importance of this because no models have been available that separate vascular cyclooxygenase-1 products from those generated elsewhere. Here, we have used unique endothelial-specific cyclooxygenase-1 knockout mice to show that endothelial cyclooxygenase-1 produces both protective and pathological products. Functionally, however, the overall effect of these was to drive pathological responses in the context of both vasoconstriction in vitro and the development of atherosclerosis and vascular inflammation in vivo. These data provide the first demonstration of a pathological role for the vascular cyclooxygenase-1 pathway, highlighting its potential as a therapeutic target. They also emphasize that, across biology, the role of prostanoids is not always predictable due to unique balances of context, products, and receptors.

An acute exposure to intermittent negative airway pressure elicits respiratory long-term facilitation in awake humans.

BACKGROUND: A sustained elevation in respiratory drive following removal of the inducing stimulus is known as respiratory long-term facilitation (rLTF). We investigated whether an acute exposure to intermittent negative airway pressure (INAP) elicits rLTF in humans. METHOD: 13 healthy males (20.9 ±â€¯2.8 years) undertook two trials (INAP and Control). In the INAP trial participants were exposed to one hour of 30-second episodes of breathing against negative pressure (-10 cmH2O) interspersed by 60-second intervals of breathing at atmospheric pressure. In the Control trial participants breathed at atmospheric pressure for one hour. Ventilation following INAP (recovery phase) was compared to that during baseline. RESULTS: Ventilation increased from baseline to recovery in the INAP trial (14.9 ±â€¯0.9 vs 19.1 ±â€¯0.7 L/min, P = 0.002). This increase was significantly greater than the equivalent during the Control trial (P = 0.019). Data shown as mean ± SEM. CONCLUSION: In this study INAP elicited rLTF in awake, healthy humans. Further research is required to investigate the responsible mechanisms.

Worming our way to novel drug discovery with the Caenorhabditis elegans proteostasis network, stress response and insulin-signaling pathways.

INTRODUCTION: Many human diseases result from a failure of a single protein to achieve the correct folding and tertiary conformation. These so-called 'conformational diseases' involve diverse proteins and distinctive cellular pathologies. They all engage the proteostasis network (PN), to varying degrees in an attempt to mange cellular stress and restore protein homeostasis. The insulin/insulin-like growth factor signaling (IIS) pathway is a master regulator of cellular stress response, which is implicated in regulating components of the PN. AREAS COVERED: This review focuses on novel approaches to target conformational diseases. The authors discuss the evidence supporting the involvement of the IIS pathway in modulating the PN and regulating proteostasis in Caenorhabditis elegans. Furthermore, they review previous PN and IIS drug screens and explore the possibility of using C. elegans for whole organism-based drug discovery for modulators of IIS-proteostasis pathways. EXPERT OPINION: An alternative approach to develop individualized therapy for each conformational disease is to modulate the global PN. The involvement of the IIS pathway in regulating longevity and response to a variety of stresses is well documented. Increasing data now provide evidence for the close association between the IIS and the PN pathways. The authors believe that high-throughput screening campaigns, which target the C. elegans IIS pathway, may identify drugs that are efficacious in treating numerous conformational diseases.

Survey Regarding Provisions of a 3-Day Supply of an Antihypertensive.

OBJECTIVE: To evaluate the provisions made by pharmacists when dispensing an emergency supply of an antihypertensive medication to patients in a community setting. PARTICIPANTS: Pharmacists and nonpharmacists (pharmacy technicians or interns) who were employed with community pharmacies and have witnessed or dispensed an emergency supply of an antihypertensive medication. Those who agreed to participate in this study via informed consent. INTERVENTION: A short questionnaire was used to assess the provisions made by community pharmacists from the perspectives of both the pharmacists and the nonpharmacists. MAIN OUTCOME MEASURES: Availability of blood pressure machines, evaluation of blood pressure readings, and patient counseling sessions or assessments prior to dispensing the emergency supply of an antihypertensive were the major outcome measures. RESULTS: Among the participants, 92% of the pharmacists and 79% of the nonpharmacists reported they have witnessed or dispensed an emergency supply of an antihypertensive medication. Of those, 82% of the pharmacists and 78% of the nonpharmacists recognized there were blood pressure machines available. However, 78% of the pharmacists and 72% of the nonpharmacists acknowledged the patient's blood pressure was not checked. CONCLUSION/IMPLICATION: This observational study demonstrates that provisions when dispensing an emergency supply of an antihypertensive medication are inconsistent. Further evaluation is warranted.

Fluphenazine reduces proteotoxicity in C. elegans and mammalian models of alpha-1-antitrypsin deficiency.

The classical form of α1-antitrypsin deficiency (ATD) is associated with hepatic fibrosis and hepatocellular carcinoma. It is caused by the proteotoxic effect of a mutant secretory protein that aberrantly accumulates in the endoplasmic reticulum of liver cells. Recently we developed a model of this deficiency in C. elegans and adapted it for high-content drug screening using an automated, image-based array scanning. Screening of the Library of Pharmacologically Active Compounds identified fluphenazine (Flu) among several other compounds as a drug which reduced intracellular accumulation of mutant α1-antitrypsin Z (ATZ). Because it is representative of the phenothiazine drug class that appears to have autophagy enhancer properties in addition to mood stabilizing activity, and can be relatively easily re-purposed, we further investigated its effects on mutant ATZ. The results indicate that Flu reverses the phenotypic effects of ATZ accumulation in the C. elegans model of ATD at doses which increase the number of autophagosomes in vivo. Furthermore, in nanomolar concentrations, Flu enhances the rate of intracellular degradation of ATZ and reduces the cellular ATZ load in mammalian cell line models. In the PiZ mouse model Flu reduces the accumulation of ATZ in the liver and mediates a decrease in hepatic fibrosis. These results show that Flu can reduce the proteotoxicity of ATZ accumulation in vivo and, because it has been used safely in humans, this drug can be moved rapidly into trials for liver disease due to ATD. The results also provide further validation for drug discovery using C. elegans models that can be adapted to high-content drug screening platforms and used together with mammalian cell line and animal models.