A randomised, blinded, placebo-controlled trial in dementia patients continuing or stopping neuroleptics (the DART-AD trial)

Background

There have been increasing concerns regarding the safety and efficacy of neuroleptics in people with dementia, but there are very few long-term trials to inform clinical practice. The aim of this study was to determine the impact of long-term treatment with neuroleptic agents upon global cognitive decline and neuropsychiatric symptoms in patients with Alzheimer disease.

Methods and Findings

Design: Randomised, blinded, placebo-controlled parallel two-group treatment discontinuation trial.Setting: Oxfordshire, Newcastle and Gateshead, London and Edinburgh, United Kingdom.Participants: Patients currently prescribed the neuroleptics thioridazine, chlorpromazine, haloperidol trifluoperazine or risperidone for behavioural or psychiatric disturbance in dementia for at least 3 mo.Interventions: Continue neuroleptic treatment for 12 mo or switch to an identical placebo.Outcome measures: Primary outcome was total Severe Impairment Battery (SIB) score. Neuropsychiatric symptoms were evaluated with the Neuropsychiatric Inventory (NPI).Results: 165 patients were randomised (83 to continue treatment and 82 to placebo, i.e., discontinue treatment), of whom 128 (78%) commenced treatment (64 continue/64 placebo). Of those, 26 were lost to follow-up (13 per arm), resulting in 51 patients per arm analysed for the primary outcome. There was no significant difference between the continue treatment and placebo groups in the estimated mean change in SIB scores between baseline and 6 mo; estimated mean difference in deterioration (favouring placebo) −0.4 (95% confidence interval [CI] −6.4 to 5.5), adjusted for baseline value (p = 0.9). For neuropsychiatric symptoms, there was no significant difference between the continue treatment and placebo groups (n = 56 and 53, respectively) in the estimated mean change in NPI scores between baseline and 6 mo; estimated mean difference in deterioration (favouring continue treatment) −2.4 (95% CI −8.2 to 3.5), adjusted for baseline value (p = 0.4). Both results became more pronounced at 12 mo. There was some evidence to suggest that those patients with initial NPI ≥ 15 benefited on neuropsychiatric symptoms from continuing treatment.

Conclusions

For most patients with AD, withdrawal of neuroleptics had no overall detrimental effect on functional and cognitive status. Neuroleptics may have some value in the maintenance treatment of more severe neuropsychiatric symptoms, but this benefit must be weighed against the side effects of therapy.Trial registration: Cochrane Central Registry of Controlled Trials/National Research Register (#ISRCTN33368770).     

Pyrrolo[1,2-b]pyridazin-2-ones as potent inhibitors of HCV NS5B polymerase

Pyrrolo[1,2-b]pyridazin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Structure-based design led to the discovery of compound 3 k, which displayed potent inhibitory activities in biochemical and replicon assays (IC(50) (1b)<10nM; EC(50) (1b)=12 nM) as well as good stability towards human liver microsomes (HLM t(1/2)>60 min).     

4-(1,1-Dioxo-1,4-dihydro-1lambda6-benzo[1,4]thiazin-3-yl)-5-hydroxy-2H-pyridazin-3-ones as potent inhibitors of HCV NS5B polymerase

4-(1,1-Dioxo-1,4-dihydro-1lambda(6)-benzo[1,4]thiazin-3-yl)-5-hydroxy-2H-pyridazin-3-one analogs were discovered as a novel class of inhibitors of HCV NS5B polymerase. Structure-based design led to the identification of compound 3a that displayed potent inhibitory activities in biochemical and replicon assays (1b IC(50)<10 nM; 1b EC(50)=1.1 nM) as well as good stability toward human liver microsomes (HLM t(1/2)>60 min).     

The O-Hyp glycosylation code in tobacco and Arabidopsis and a proposed role of Hyp-glycans in secretion

Most aspects of plant growth involve cell surface hydroxyproline (Hyp)-rich glycoproteins (HRGPs) whose properties depend on arabinogalactan polysaccharides and arabinosides that define the molecular surface. Potential glycosylation sites are defined by an O-Hyp glycosylation code: contiguous Hyp directs arabinosylation. Clustered non-contiguous Hyp directs arabinogalactosylation. Elucidation of this code involved a single species, tobacco (Nicotiana tabacum) BY-2 cells. However, recent work suggests species variation, perhaps tissue specific Hyp glycosylation. Thus, the extent to which the Hyp glycosylation code is 'global' needs testing. We compared the ability of distantly related Arabidopsis cell cultures to process putative HRGP glycosylation motifs encoded by synthetic genes. The genes included: repetitive Ser-Pro, Ser-Pro2, Ser-Pro4 and an analog of the tomato arabinogalactan-protein, LeAGP-1DeltaGPI. All were expressed as enhanced green fluorescent protein (EGFP) fusion glycoproteins, designated: AtSO-EGFP (O=Hyp), AtSO2-EGFP, AtSO4-EGFP and AtEGFP-LeAGP-1DeltaGPI, respectively. The Arabidopsis glycosylation patterns were essentially similar to those observed in Nicotiana: non-contiguous Hyp residues in AtSO-EGFP were glycosylated exclusively with arabinogalactan polysaccharides while contiguous Hyp in AtSO2-EGFP and AtSO4-EGFP was exclusively arabinosylated. Mixed contiguous and non-contiguous Hyp residues in AtEGFP-LeAGP-1DeltaGPI were also arabinosylated and arabinogalactosylated consistent with the code. However, slightly more arabinogalactosylated Hyp and less non-glycosylated Hyp in AtEGFP-LeAGP-1DeltaGPI than tobacco NtEGFP-LeAGP-1DeltaGPI suggested Arabidopsis prolyl hydroxylases have a slightly broader specificity. Arabidopsis Hyp-arabinogalactans differed from tobacco in decreased glucuronic acid content and lack of rhamnose. Yields of the EGFP fusion glycoproteins were dramatically higher than targeted EGFP lacking Hyp-glycomodules. This validates earlier suggestions that the glycosylation of proteins facilitates their secretion.     

AtAGP18 is localized at the plasma membrane and functions in plant growth and development

Arabinogalactan-proteins (AGPs) are a family of highly glycosylated hydroxyproline-rich glycoproteins (HRGPs). AtAGP17, 18 and 19 comprise the lysine-rich classical AGP subfamily in Arabidopsis. Overexpression of GFP–AtAGP17/18/19 fusion proteins in Arabidopsis revealed localization of the fusion proteins on the plant cell surface of different organs. Subcellular localization of the fusion proteins at the plasma membrane was further determined by plasmolysis of leaf trichome cells. To elucidate AtAGP17/18/19 function(s), these AGPs were expressed without the green fluorescent protein (GFP) tag under the control of 35S cauliflower mosaic virus promoter. In contrast to AtAGP17/AtAGP19 overexpressors which showed phenotypes identical to wild-type plants, AtAGP18 overexpressors displayed several phenotypes distinct from wild-type plants. Specifically, these overexpressors had smaller rosettes and shorter stems and roots, produced more branches and had less viable seeds. Moreover, these AtAGP18 overexpressors exhibited similar phenotypes to tomato LeAGP-1 overexpressors, suggesting these two AGP genes may have similar function(s) in Arabidopsis and tomato.     

Progesterone Receptor Membrane Component 1 Is a Functional Part of the Glucagon-like Peptide-1 (GLP-1) Receptor Complex in Pancreatic β Cells

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that regulates glucose homeostasis. Because of their direct stimulation of insulin secretion from pancreatic β cells, GLP-1 receptor (GLP-1R) agonists are now important therapeutic options for the treatment of type 2 diabetes. To better understand the mechanisms that control the insulinotropic actions of GLP-1, affinity purification and mass spectrometry (AP-MS) were employed to uncover potential proteins that functionally interact with the GLP-1R. AP-MS performed on Chinese hamster ovary cells or MIN6 β cells, both expressing the human GLP-1R, revealed 99 proteins potentially associated with the GLP-1R. Three novel GLP-1R interactors (PGRMC1, Rab5b, and Rab5c) were further validated through co-immunoprecipitation/immunoblotting, fluorescence resonance energy transfer, and immunofluorescence. Functional studies revealed that overexpression of PGRMC1, a novel cell surface receptor that associated with liganded GLP-1R, enhanced GLP-1-induced insulin secretion (GIIS) with the most robust effect. Knockdown of PGRMC1 in β cells decreased GIIS, indicative of positive interaction with GLP-1R. To gain insight mechanistically, we demonstrated that the cell surface PGRMC1 ligand P4-BSA increased GIIS, whereas its antagonist AG-205 decreased GIIS. It was then found that PGRMC1 increased GLP-1-induced cAMP accumulation. PGRMC1 activation and GIIS induced by P4-BSA could be blocked by inhibition of adenylyl cyclase/EPAC signaling or the EGF receptor–PI3K signal transduction pathway. These data reveal a dual mechanism for PGRMC1-increased GIIS mediated through cAMP and EGF receptor signaling. In conclusion, we identified several novel GLP-1R interacting proteins. PGRMC1 expressed on the cell surface of β cells was shown to interact with the activated GLP-1R to enhance the insulinotropic actions of GLP-1.Glucagon-like peptide-1 (GLP-1)¹ is a gastrointestinal hormone secreted by intestinal L cells upon food intake that is best known for its role in controlling glucose homeostasis. Acting through its cognate glucagon-like peptide-1 receptor (GLP-1R), GLP-1 has several important physiological and pharmacological functions. GLP-1 is best known for enhancing glucose-stimulated insulin secretion (GSIS) from the pancreatic β cells. Importantly, the insulinotropic properties of GLP-1 are maintained in patients with type 2 diabetes (1), which is characterized by insufficient insulin secretion from pancreatic β cells and an inability to maintain glucose homeostasis. Therefore, therapeutic strategies targeting GLP-1R have been developed to treat type 2 diabetes (2, 3). In addition to augmenting insulin secretion, GLP-1 has been known to improve glucose sensing, proinsulin biosynthesis, survival, and proliferation of β cells (3, 4) in a variety of experimental models. GLP-1 also has several extrapancreatic effects, including actions on the central nervous system to inhibit food intake (5), the stomach to decrease gastric emptying and gastric acid secretion (6), and the lungs to stimulate secretion of macromolecules from airways (7). Additionally, GLP-1 has an effect on the heart and possibly the kidney to modulate blood pressure and heart rate (8, 9).The GLP-1R is a member of the B1 family of G protein–coupled receptors (secretin receptor family). In mammals, GLP-1R is expressed in multiple tissues, including pancreatic β cells and δ cells (10), hypothalamus, lung, stomach, heart, kidney (11), and thyroid (12), which in part explains its diverse actions. Upon ligand binding, the GLP-1R is capable of coupling to diverse cell signal transduction pathways, but it is best known for its actions on G protein Gs α and adenylate cyclase activity to increase intracellular cAMP. It is known that other proteins can affect GLP-1R activity in addition to G proteins, including β-arrestin and caveolin, which affect receptor internalization and trafficking. β-Arrestin 1 is also required for proper GLP-1-stimulated cAMP production (13–15). More recently, it was shown that another B1 family member, gastric inhibitory polypeptide receptor heterodimerizes with GLP-1R, decreasing GLP-1-induced β-arrestin recruitment and mobilization (16). Very recently, our group identified several novel potential GLP-1R interactors using a membrane-based split-ubiquitin yeast two-hybrid (MYTH) assay (17). Three β cell–expressing membrane-bound interactors, solute carrier family 15 member 4 (SLC15A4), amyloid β A4 precursor-like protein 1 (APLP1), and adaptor-related protein complex 2 subunit mu (AP2M1), were further selected for individual knockdown in mouse insulinoma (MIN6) β cells using small interfering RNAs (siRNAs). GLP-1-induced insulin secretion was significantly enhanced when these genes were silenced, suggesting that these interactor proteins attenuate GLP-1R activity. These findings demonstrated that GLP-1R protein interactions are complex and the interactors can have measurable effects on receptor trafficking and downstream signaling. Such interactions may in part explain the diverse tissue-specific effects of GLP-1 and offer avenues for controlling GLP-1 actions in a tissue-selective manner.Although the MYTH system is well established (18) and has been applied to study G protein–coupled receptor interactomes (17), it is limited on two fronts. Firstly, it must be performed in yeast which is not an ideal representation of the mammalian system. Secondly, it is technically difficult to activate the receptor in MYTH, thus, effects of ligand stimulation on the receptor interactome cannot be assessed. Recently, affinity purification–mass spectrometry (AP-MS) has become a powerful tool for discovering and examining novel protein–protein interactions, including those between membrane-bound proteins in mammalian cells (19–21). In the current study, we applied AP-MS to discover novel GLP-1R interactors and employed a human GLP-1R harboring a FLAG^® epitope. GLP-1R-Flag was expressed in either Chinese hamster ovary (CHO) cells or MIN6 β cells, and interactors were studied in the presence or absence of GLP-1.     

Molecular details of tomato extensin and glycine-rich protein gene expression

In a recent publication (Plant Molecular Biology 16: 547–565 (1991)) Showalter et al. described the isolation and initial characterization of fifteen extensin and extensin-like tomato cDNAs. These cDNAs were determined to fall into five distinct classes; class I and II clones encoded extensins, class III and V clones encoded glycine-rich proteins (GRPs), and class IV clones encoded a portion of a GRP sequence on one DNA strand and a portion of an extensin sequence on the other DNA strand. In this publication, a more detailed analysis of the expression of these cDNA classes was performed with respect to wounding in various tomato organs, development, kinetics and systemic extent of the wound response, ethylene treatment, abscisic acid (ABA) treatment, and drought stress by using RNA gel blot hybridizations. In general, extensin gene expression was readily detected in stems and roots, but not in leaves. With both class I and II extensin cDNA probes, wound-induced accumulation of mRNA in stems was first detected between 4 and 8 h after wounding with maximal accumulation occurring after 12 h. Moreover, these extensin wound responses were detected locally at the wound site but not systemically. Expression of the class III GRP was largely limited to wounded stem tissue. Initial detection and maximal accumulation of the class III GRP mRNA was similar to the extensins mRNAs; however, this GRP wound response occurred both locally and systemically. Additionally, abscisic acid treatment and drought stress resulted in the marked accumulation of the class III GRP mRNA in tomato stems, but did not alter the expression of the other cDNA classes. In contrast, expression of the class V GRP occurred in stems and roots and to a lesser extent in leaves and decreased in response to wounding over a 24 h time period. The class V GRP wound response was further characterized by an early, transient accumulation of mRNA occurring 2–4 h after wounding in stems and by its local nature.     

Glycosylation of a Fasciclin-Like Arabinogalactan-Protein (SOS5) Mediates Root Growth and Seed Mucilage Adherence via a Cell Wall Receptor-Like Kinase (FEI1/FEI2) Pathway in Arabidopsis

Fundamental processes that underpin plant growth and development depend crucially on the action and assembly of the cell wall, a dynamic structure that changes in response to both developmental and environmental cues. While much is known about cell wall structure and biosynthesis, much less is known about the functions of the individual wall components, particularly with respect to their potential roles in cellular signaling. Loss-of-function mutants of two arabinogalactan-protein (AGP)-specific galactosyltransferases namely, GALT2 and GALT5, confer pleiotropic growth and development phenotypes indicating the important contributions of carbohydrate moieties towards AGP function. Notably, galt2galt5 double mutants displayed impaired root growth and root tip swelling in response to salt, likely as a result of decreased cellulose synthesis. These mutants phenocopy a salt-overly sensitive mutant called sos5, which lacks a fasciclin-like AGP (SOS5/FLA4) as well as a fei1fei2 double mutant, which lacks two cell wall-associated leucine-rich repeat receptor-like kinases. Additionally, galt2gal5 as well as sos5 and fei2 showed reduced seed mucilage adherence. Quintuple galt2galt5sos5fei1fei2 mutants were produced and provided evidence that these genes act in a single, linear genetic pathway. Further genetic and biochemical analysis of the quintuple mutant demonstrated involvement of these genes with the interplay between cellulose biosynthesis and two plant growth regulators, ethylene and ABA, in modulating root cell wall integrity.