Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p<0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (<1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (<1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

Structural Plasticity of Dendritic Spines Requires GSK3α and GSK3β

<div><p>Although memories appear to be elusive phenomena, they are stored in the network of physical connections between neurons. Dendritic spines, which are actin-rich dendritic protrusions, serve as the contact points between networked neurons. The spines’ shape contributes to the strength of signal transmission. To acquire and store information, dendritic spines must remain plastic, i.e., able to respond to signals, by changing their shape. We asked whether glycogen synthase kinase (GSK) 3α and GSK3β, which are implicated in diseases with neuropsychiatric symptoms, such as Alzheimer's disease, bipolar disease and schizophrenia, play a role in a spine structural plasticity. We used Latrunculin B, an actin polymerization inhibitor, and chemically induced Long-Term Depression to trigger fast spine shape remodeling in cultured hippocampal neurons. Spine shrinkage induced by either stimulus required GSK3α activity. GSK3β activity was only important for spine structural changes after treatment with Latrunculin B. Our results indicate that GSK3α is an essential component for short-term spine structural plasticity. This specific function should be considered in future studies of neurodegenerative diseases and neuropsychiatric conditions that originate from suboptimal levels of GSK3α/β activity.</p></div

Antibodies used in the study.

<p>Antibodies used in the study.</p

GSK3α and GSK3β knockdown alter LatrB-induced changes to dendritic spine morphology.

<p>A. GSK3 α/β knockdown does not affect basal fluctuations of dendritic spine morphology. Experimental outline with 4 time points for microscopy and quantitative analysis of spine shape; # indicates <i>p</i><0.05 for measurements of spines after GSK3β silencing compared to control values at the corresponding time points. For number of counted spines refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134018#pone.0134018.t003" target="_blank">Table 3</a>. Data are presented as the mean spine width per cell ± s.e.m. The curve between time points is extrapolated. B. Experimental outline with 3 time points for microscopy: baseline, LatrB treatment, end of recovery period. Representative micrographs of cultured DIV18 murine hippocampal neurons transfected with shRNA constructs as indicated. Scale bar = 2.5 μm. C. Quantitative analysis of spine shape changes; *** = <i>p</i> < 0.001 and xx = <i>p</i> < 0.01, xxx = <i>p</i> < 0.001 and ### = <i>p</i> < 0.001 for spine measurements of shRNA silenced GSK3α, GSK3β and GSK3α/β compared to the control at the corresponding time points. ‡ = <i>p</i> < 0.05 difference between shRNAGSK3α and shGSK3β. For number of counted spines refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134018#pone.0134018.t003" target="_blank">Table 3</a>. Data are presented as mean spine width per cell ± s.e.m. The curve between time points is extrapolated. D. Spine l/w ratio changes presented as cumulative histograms of length/width ratio at 3 time points.</p

Knockdown of GSK3α but not GSK3β affects chLTD-induced changes to dendritic spine morphology

<p>A. Experimental outline with 3 time points for microscopy: baseline, chLTD induction, end of recovery period. Representative micrographs of cultured DIV18 murine hippocampal neurons transfected with shRNA constructs as indicated. Scale bar = 2.5 μm. B. Quantitative analysis of spine shape changes; * = <i>p</i> < 0.05, ** = <i>p</i> < 0.01 and x = <i>p</i> <0.05 and xxx = <i>p</i> < 0.001 for measurements of spines after GSK3α and GSK3α/β silencing compared to the control at the corresponding time points. ‡ = <i>p</i> < 0.05 difference between GSK3α and GSK3α/β silencing. For number of counted spines refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134018#pone.0134018.t003" target="_blank">Table 3</a>. Data are presented as the mean spine width per cell ± s.e.m. The curve between time points is extrapolated. C. Spine l/w ratio changes are presented as cumulative histograms of the l/w ratio at 3 time points.</p

Inhibition of GSK3α/β activity in cultured neurons treated with LatrB hinders fast spine structural changes.

<p>A. Efficiency of GSK3α/β chemical inhibition. The level of phosphorylation for glycogen synthase Ser 641 at time points after GSK3α/β inhibition with Ch98 and BIO. Lysates from hippocampal neurons on DIV18. Tubulin was the loading control. B. Pharmacological inhibition of GSK3α/β does not affect basal fluctuations of dendritic spine morphology. Experimental outline with 4 time points for microscopy and quantitative analysis of spine width; ## indicates <i>p</i><0.01 for measurements of spines after GSK3α/β inhibition with BIO compared with control values at the corresponding time point. For number of counted spines refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134018#pone.0134018.t003" target="_blank">Table 3</a>. Data are presented as the mean spine width per cell ± s.e.m. The curve between time points is extrapolated. C. Experimental outline with 3 time points for microscopy: baseline, LatrB treatment, end of recovery period. Representative micrographs of DIV18 cultured murine hippocampal neurons. Scale bar = 2.5 μm. D. Quantitative analysis of spine shape changes; *** and ### indicates <i>p</i><0.001 for measurements of spines after GSK3α/β inhibition by Ch98 and BIO when compared to control values at the corresponding time points. For number of counted spines refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134018#pone.0134018.t003" target="_blank">Table 3</a>. Data are presented as mean spine width per cell ± s.e.m. The curve between time points is extrapolated. E. Spine l/w ratio changes are presented as cumulative histograms of the l/w ratio at 3 time points.</p

Inhibition of GSK3α/β activity in cultured neurons hinders spine structural plasticity upon chLTD

<p>A. chLTD activates GSK3α/β in synaptoneurosomes isolated from murine hippocampi. Representative immunoblots, scanned with a Li-Cor Odyssey imager, for phospho-GSK3α (Ser21)/β (Ser9), total GSK3α/β, phospho-GluA1 (Ser845; LTD control) and GAPDH (loading control). B. Quantitative analysis of phospho-GSK3α/β to the total-GSK3α/β ratio at the time points indicated, expressed as % values of an untreated control. Data (n = 3 experiments) are presented as means ± s.e.m. * indicates a <i>p</i><0.05 vs control. C. Pharmacological inhibition of GSK3α/β affects chemical LTD-induced changes of dendritic spine width. Experimental outline with 3 time points for microscopy: baseline, chLTD induction, end of recovery period. Representative micrographs of DIV18 cultured murine hippocampal neurons. Scale bar = 2.5 μm. D. Quantitative analysis of spine shape changes; *** = <i>p</i> < 0.001, # = <i>p</i> < 0.05, ## = <i>p</i> <0.01 for spine measurements after GSK3α/β inhibition with Ch98 and BIO compared to control values at the corresponding time points. For number of counted spines refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134018#pone.0134018.t003" target="_blank">Table 3</a>. Data are presented as mean spine width per cell ± s.e.m. The curve between time points is extrapolated. E. Spine l/w ratio changes are presented as cumulative histograms of the length/width ratio at 3 time points.</p

Validation of GSK3 shRNAs and their scrambled counterparts.

<p>A. Representative micrographs of neurons transfected with shRNA constructs (as indicated) and immunofluorescently labeled for GSK3α or GSK3β. Scale bar = 50 μm. B. Quantitative analysis of GSK3 silencing; * = <i>p</i> < 0.05, ** = <i>p</i> <0.01 and *** = <i>p</i> < 0.001 for intensity measurements compared to neurons expressing pSuper^GFP. ### = <i>p</i> < 0.001 for experimental variants indicated by brackets. Data are presented as the mean ± s.e.m.</p

Impaired synaptic incorporation of AMPA receptors in a mouse model of fragile X syndrome

Fragile X syndrome (FXS) is the most common monogenetic cause of inherited intellectual disability and autism in humans. One of the well-characterized molecular phenotypes of Fmr1 KO mice, a model of FXS, is increased translation of synaptic proteins. Although this upregulation stabilizes in adulthood, abnormalities during the critical period of plasticity have long-term effects on circuit formation and synaptic properties. Using high-resolution quantitative proteomics of synaptoneurosomes isolated from the adult, developed brains of Fmr1 KO mice, we show a differential abundance of proteins regulating the postsynaptic receptor activity of glutamatergic synapses. We investigated the AMPA receptor composition and shuttling in adult Fmr1 KO and WT mice using a variety of complementary experimental strategies such as surface protein crosslinking, immunostaining of surface receptors, and electrophysiology. We discovered that the activity-dependent synaptic delivery of AMPARs is impaired in adult Fmr1 KO mice. Furthermore, we show that Fmr1 KO synaptic AMPARs contain more GluA2 subunits that can be interpreted as a switch in the synaptic AMPAR subtype toward an increased number of Ca2+−impermeable receptors in adult Fmr1 KO synapses

TIAR and FMRP shape pro-survival nascent proteome of leukemia cells in the bone marrow microenvironment

Summary: Chronic myeloid leukemia (CML) cells circulate between blood and bone marrow niche, representing different microenvironments. We studied the role of the two RNA-binding proteins, T-cell-restricted intracellular antigen (TIAR), and the fragile X mental retardation protein (FMRP) in the regulation of protein translation in CML cells residing in settings mimicking peripheral blood microenvironment (PBM) and bone marrow microenvironment (BMM). The outcomes showed how conditions shaped the translation process through TIAR and FMRP activity, considering its relevance in therapy resistance. The QuaNCAT mass-spectrometric approach revealed that TIAR and FMRP have a discrete modulatory effect on protein synthesis and thus affect distinct aspects of leukemic cells functioning in the hypoxic niche. In the BMM setup, FMRP impacted metabolic adaptation of cells and TIAR substantially supported the resistance of CML cells to translation inhibition by homoharringtonine. Overall, our results demonstrated that targeting post-transcriptional control should be considered when designing anti-leukemia therapeutic solutions