Catharine A. Ortori

Oral Treatment Targeting the Unfolded Protein Response Prevents Neurodegeneration and Clinical Disease in Prion-Infected Mice

Pharmacological inhibition of PERK, the key kinase of the unfolded protein response that mediates translational shutdown, restores protein synthesis in prion-infected mice, thus preventing neurodegeneration and clinical disease. Perking Up Prion Disease Therapy There are no effective treatments for neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and prion disease. These diseases share common features, including the accumulation of misfolded disease-specific proteins in the brain, leading to neuronal loss, which is ultimately fatal. In addition, the brains of patients with these neurodegenerative diseases show overactivation of a cellular defense pathway for dealing with misfolded proteins called the unfolded protein response (UPR). The UPR deals with the misfolded protein load in a number of ways including transiently switching off translation. Moreno et al. now report that the buildup of misfolded prion protein in mice with prion disease causes sustained overactivation of this pathway. This results in long-term translational inhibition, causing a critical decline in key proteins needed for neuronal survival. The authors used a newly described specific inhibitor of a key UPR kinase mediating translational shutdown to test if pharmacological inhibition would be neuroprotective. The compound prevented neurodegeneration and the emergence of clinical disease in prion-infected mice, whereas untreated animals all succumbed to disease. These data suggest that the UPR may represent a new therapeutic target for drug development to treat prion disease and possibly other neurodegenerative diseases as well. During prion disease, an increase in misfolded prion protein (PrP) generated by prion replication leads to sustained overactivation of the branch of the unfolded protein response (UPR) that controls the initiation of protein synthesis. This results in persistent repression of translation, resulting in the loss of critical proteins that leads to synaptic failure and neuronal death. We have previously reported that localized genetic manipulation of this pathway rescues shutdown of translation and prevents neurodegeneration in a mouse model of prion disease, suggesting that pharmacological inhibition of this pathway might be of therapeutic benefit. We show that oral treatment with a specific inhibitor of the kinase PERK (protein kinase RNA–like endoplasmic reticulum kinase), a key mediator of this UPR pathway, prevented UPR-mediated translational repression and abrogated development of clinical prion disease in mice, with neuroprotection observed throughout the mouse brain. This was the case for animals treated both at the preclinical stage and also later in disease when behavioral signs had emerged. Critically, the compound acts downstream and independently of the primary pathogenic process of prion replication and is effective despite continuing accumulation of misfolded PrP. These data suggest that PERK, and other members of this pathway, may be new therapeutic targets for developing drugs against prion disease or other neurodegenerative diseases where the UPR has been implicated.

Broccoli Consumption Interacts with GSTM1 to Perturb Oncogenic Signalling Pathways in the Prostate

Background Epidemiological studies suggest that people who consume more than one portion of cruciferous vegetables per week are at lower risk of both the incidence of prostate cancer and of developing aggressive prostate cancer but there is little understanding of the underlying mechanisms. In this study, we quantify and interpret changes in global gene expression patterns in the human prostate gland before, during and after a 12 month broccoli-rich diet. Methods and Findings Volunteers were randomly assigned to either a broccoli-rich or a pea-rich diet. After six months there were no differences in gene expression between glutathione S-transferase mu 1 (GSTM1) positive and null individuals on the pea-rich diet but significant differences between GSTM1 genotypes on the broccoli-rich diet, associated with transforming growth factor beta 1 (TGFβ1) and epidermal growth factor (EGF) signalling pathways. Comparison of biopsies obtained pre and post intervention revealed more changes in gene expression occurred in individuals on a broccoli-rich diet than in those on a pea-rich diet. While there were changes in androgen signalling, regardless of diet, men on the broccoli diet had additional changes to mRNA processing, and TGFβ1, EGF and insulin signalling. We also provide evidence that sulforaphane (the isothiocyanate derived from 4-methylsuphinylbutyl glucosinolate that accumulates in broccoli) chemically interacts with TGFβ1, EGF and insulin peptides to form thioureas, and enhances TGFβ1/Smad-mediated transcription. Conclusions These findings suggest that consuming broccoli interacts with GSTM1 genotype to result in complex changes to signalling pathways associated with inflammation and carcinogenesis in the prostate. We propose that these changes may be mediated through the chemical interaction of isothiocyanates with signalling peptides in the plasma. This study provides, for the first time, experimental evidence obtained in humans to support observational studies that diets rich in cruciferous vegetables may reduce the risk of prostate cancer and other chronic disease. Trial Registration ClinicalTrials.gov NCT00535977

Simultaneous quantitative profiling of N-acyl-l-homoserine lactone and 2-alkyl-4(1H)-quinolone families of quorum-sensing signaling molecules using LC-MS/MS

An LC-MS/MS method, using positive mode electrospray ionization, for the simultaneous, quantitative and targeted profiling of the N-acyl-l-homoserine lactone (AHL) and 2-alkyl 4-(1H)-quinolone (AQ) families of bacterial quorum-sensing signaling molecules (QSSMs) is presented. This LC-MS/MS technique was applied to determine the relative molar ratios of AHLs and AQs produced by Pseudomonas aeruginosa and the consequences of mutating individual or multiple QSSM synthase genes (lasI, rhlI, pqsA) on AHL and AQ profiles and concentrations. The AHL profile of P. aeruginosa was dominated by N-butanoyl-l-homoserine lactone (C4-HSL) with lesser concentrations of N-hexanoyl-l-homoserine lactone (C6-HSL) and 3-oxo-substituted longer chain AHLs including N-(3-oxodecanoyl)-l-homoserine lactone (3-oxo-C10-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL). The AQ profile of P. aeruginosa comprised the C7 and C9 long alkyl chain AQs including 2-heptyl-4-hydroxyquinoline (HHQ), 2-nonyl-4-hydroxyquinoline, the “pseudomonas quinolone signal” (2-heptyl-3-hydroxy-4-quinolone) and the N-oxides, 2-heptyl-4-hydroxyquinoline N-oxide and 2-nonyl-4-hydroxyquinoline N-oxide. Application of the method showed significant effects of growth medium type on the ratio and the nature of the QSSMs synthesized and the dramatic effect of single, double and triple mutations in the P. aeruginosa QS synthase genes. The LC-MS/MS methodology is applicable in organisms where either or both AHL and AQ QSSMs are produced and can provide comprehensive profiles and concentrations from a single sample.

Partial restoration of protein synthesis rates by the small molecule ISRIB prevents neurodegeneration without pancreatic toxicity

Activation of the PERK branch of the unfolded protein response (UPR) in response to protein misfolding within the endoplasmic reticulum (ER) results in the transient repression of protein synthesis, mediated by the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α). This is part of a wider integrated physiological response to maintain proteostasis in the face of ER stress, the dysregulation of which is increasingly associated with a wide range of diseases, particularly neurodegenerative disorders. In prion-diseased mice, persistently high levels of eIF2α cause sustained translational repression leading to catastrophic reduction of critical proteins, resulting in synaptic failure and neuronal loss. We previously showed that restoration of global protein synthesis using the PERK inhibitor GSK2606414 was profoundly neuroprotective, preventing clinical disease in prion-infected mice. However, this occured at the cost of toxicity to secretory tissue, where UPR activation is essential to healthy functioning. Here we show that pharmacological modulation of eIF2α-P-mediated translational inhibition can be achieved to produce neuroprotection without pancreatic toxicity. We found that treatment with the small molecule ISRIB, which restores translation downstream of eIF2α, conferred neuroprotection in prion-diseased mice without adverse effects on the pancreas. Critically, ISRIB treatment resulted in only partial restoration of global translation rates, as compared with the complete restoration of protein synthesis seen with GSK2606414. ISRIB likely provides sufficient rates of protein synthesis for neuronal survival, while allowing some residual protective UPR function in secretory tissue. Thus, fine-tuning the extent of UPR inhibition and subsequent translational de-repression uncouples neuroprotective effects from pancreatic toxicity. The data support the pursuit of this approach to develop new treatments for a range of neurodegenerative disorders that are currently incurable.

Potato Plants Genetically Modified to Produce N-Acylhomoserine Lactones Increase Susceptibility to Soft Rot Erwiniae

Many gram-negative bacteria employ N-acylhomoserine lactones (AHL) to regulate diverse physiological processes in concert with cell population density (quorum sensing [QS]). In the plant pathogen Erwinia carotovora, the AHL synthesized via the carI/expI genes are responsible for regulating the production of secreted plant cell wall-degrading exoenzymes and the antibiotic carbapen-3-em carboxylic acid. We have previously shown that targeting the product of an AHL synthase gene (yenI) from Yersinia enterocolitica to the chloroplasts of transgenic tobacco plants caused the synthesis in planta of the cognate AHL signaling molecules N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL), which in turn, were able to complement a carI-QS mutant. In the present study, we demonstrate that transgenic potato plants containing the yenI gene are also able to express AHL and that the presence and level of these AHL in the plant increases susceptibility to infection by E. carotovora. Susceptibility is further affected by both the bacterial level and the plant tissue under investigation.

Garlic as an inhibitor of Pseudomonas aeruginosa quorum sensing in cystic fibrosis--a pilot randomized controlled trial.

Pseudomonas aeruginosa forms biofilms in the cystic fibrosis lung. Quorum sensing (QS) controls biofilm maturation, immune evasion, antibiotic tolerance and virulence factor production. Garlic shows QS inhibitory activity in vitro and in animal models. We report the first clinical trial in man of a QS inhibitor.

Quantitative profiling of nucleotides and related phosphate-containing metabolites in cultured mammalian cells by liquid chromatography tandem electrospray mass spectrometry.

A method has been developed for the quantitative profiling of over twenty nucleotides and related phosphorylated species using ion-pair reversed-phase liquid chromatography hyphenated to negative ion tandem electrospray mass spectrometry. The influence of mobile phase pH and ion-pairing agent concentration were assessed to optimise separation and peak shapes. Full quantitative analysis was obtained for the nucleotides by reference to structurally related calibration standards. The developed method was applied to profile changes in nucleotides and related compounds in monolayer cultured Chinese hamster ovary (CHO) cells expressing the beta(2) adrenoceptor when exposed to pharmacological stimuli. These experiments demonstrate the potential of the LC-MS/MS method to detect changes in nucleotide drug targets as well as the simultaneous monitoring of levels of other nucleotides.

Isothiocyanate concentrations and interconversion of sulforaphane to erucin in human subjects after consumption of commercial frozen broccoli compared to fresh broccoli.

SCOPE Sulforaphane (a potent anticarcinogenic isothiocyanate derived from glucoraphanin) is widely considered responsible for the protective effects of broccoli consumption. Broccoli is typically purchased fresh or frozen and cooked before consumption. We compared the bioavailability and metabolism of sulforaphane from portions of lightly cooked fresh or frozen broccoli, and investigated the bioconversion of sulforaphane to erucin. METHODS AND RESULTS Eighteen healthy volunteers consumed broccoli soups produced from fresh or frozen broccoli florets that had been lightly cooked and sulforaphane thio-conjugates quantified in plasma and urine. Sulforaphane bioavailability was about tenfold higher for the soups made from fresh compared to frozen broccoli, and the reduction was shown to be due to destruction of myrosinase activity by the commercial blanching-freezing process. Sulforaphane appeared in plasma and urine in its free form and as several thio-conjugates forms. Erucin N-acetyl-cysteine conjugate was a significant urinary metabolite, and it was shown that human gut microflora can produce sulforaphane, erucin, and their nitriles from glucoraphanin. CONCLUSION The short period of blanching used to produce commercial frozen broccoli destroys myrosinase and substantially reduces sulforaphane bioavailability. Sulforaphane was converted to erucin and excreted in urine, and it was shown that human colonic flora were capable of this conversion.

Transient Exposure to Low Levels of Insecticide Affects Metabolic Networks of Honeybee Larvae

The survival of a species depends on its capacity to adjust to changing environmental conditions, and new stressors. Such new, anthropogenic stressors include the neonicotinoid class of crop-protecting agents, which have been implicated in the population declines of pollinating insects, including honeybees (Apis mellifera). The low-dose effects of these compounds on larval development and physiological responses have remained largely unknown. Over a period of 15 days, we provided syrup tainted with low levels (2 µg/L−1) of the neonicotinoid insecticide imidacloprid to beehives located in the field. We measured transcript levels by RNA sequencing and established lipid profiles using liquid chromatography coupled with mass spectrometry from worker-bee larvae of imidacloprid-exposed (IE) and unexposed, control (C) hives. Within a catalogue of 300 differentially expressed transcripts in larvae from IE hives, we detect significant enrichment of genes functioning in lipid-carbohydrate-mitochondrial metabolic networks. Myc-involved transcriptional response to exposure of this neonicotinoid is indicated by overrepresentation of E-box elements in the promoter regions of genes with altered expression. RNA levels for a cluster of genes encoding detoxifying P450 enzymes are elevated, with coordinated downregulation of genes in glycolytic and sugar-metabolising pathways. Expression of the environmentally responsive Hsp90 gene is also reduced, suggesting diminished buffering and stability of the developmental program. The multifaceted, physiological response described here may be of importance to our general understanding of pollinator health. Muscles, for instance, work at high glycolytic rates and flight performance could be impacted should low levels of this evolutionarily novel stressor likewise induce downregulation of energy metabolising genes in adult pollinators.

Repurposed drugs targeting eIF2α-P-mediated translational repression prevent neurodegeneration in mice

See Mercado and Hetz (doi:10.1093/brain/awx107) for a scientific commentary on this article. Signalling through the PERK/eIF2α-P branch of the Unfolded Protein Response is increased in many neurodegenerative diseases. Halliday et al. identify two safe compounds – one licensed – that act on this pathway and are neuroprotective in mice with neurodegeneration. These drugs can now be repurposed in clinical trials for the treatment of dementia.