Analyzing the mechanisms that facilitate the subtype-specific assembly of γ-aminobutyric acid type A receptors

Impaired inhibitory signaling underlies the pathophysiology of many neuropsychiatric and neurodevelopmental disorders including autism spectrum disorders and epilepsy. Neuronal inhibition is regulated by synaptic and extrasynaptic γ-aminobutyric acid type A receptors (GABAARs), which mediate phasic and tonic inhibition, respectively. These two GABAAR subtypes differ in their function, ligand sensitivity, and physiological properties. Importantly, they contain different α subunit isoforms: synaptic GABAARs contain the α1–3 subunits whereas extrasynaptic GABAARs contain the α4–6 subunits. While the subunit composition is critical for the distinct roles of synaptic and extrasynaptic GABAAR subtypes in inhibition, the molecular mechanism of the subtype-specific assembly has not been elucidated. To address this issue, we purified endogenous α1- and α4-containing GABAARs from adult murine forebrains and examined their subunit composition and interacting proteins using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and quantitative analysis. We found that the α1 and α4 subunits form separate populations of GABAARs and interact with distinct sets of binding proteins. We also discovered that the β3 subunit, which co-purifies with both the α1 and α4 subunits, has different levels of phosphorylation on serines 408 and 409 (S408/9) between the two receptor subtypes. To understand the role S408/9 plays in the assembly of α1- and α4-containing GABAARs, we examined the effects of S408/9A (alanine) knock-in mutation on the subunit composition of the two receptor subtypes using LC-MS/MS and quantitative analysis. We discovered that the S408/9A mutation results in the formation of novel α1α4-containing GABAARs. Moreover, in S408/9A mutants, the plasma membrane expression of the α4 subunit is increased whereas its retention in the endoplasmic reticulum is reduced. These findings suggest that S408/9 play a critical role in determining the subtype-specific assembly of GABAARs, and thus the efficacy of neuronal inhibition

A computationally inspired in-vivo approach identifies a link between amygdalar transcriptional heterogeneity, socialization and anxiety

Pharmaceutical breakthroughs for anxiety have been lackluster in the last half-century. Converging behavior and limbic molecular heterogeneity has the potential to revolutionize biomarker-driven interventions. However, current in vivo models too often deploy artificial systems including directed evolution, mutations and fear induction, which poorly mirror clinical manifestations. Here, we explore transcriptional heterogeneity of the amygdala in isogenic mice using an unbiased multidimensional computational approach that segregates intra-cohort reactions to moderate situational adversity and intersects it with high content molecular profiling. We show that while the computational approach stratifies known features of clinical anxiety including nitric oxide, opioid and corticotropin signaling, previously unrecognized druggable biomarkers emerge, such as calpain11 and scand1. Through ingenuity pathway analyses, we further describe a role for neurosteroid estradiol signaling, heat shock proteins, ubiquitin ligases and lipid metabolism. In addition, we report a remarkable behavioral pattern that maps to molecular features of anxiety in mice through counterphobic social attitudes, which manifest as increased, yet spatially distant socialization. These findings provide an unbiased approach for interrogating anxiolytics, and hint toward biomarkers underpinning behavioral and social patterns that merit further exploration

Identification of a Core Amino Acid Motif within the α Subunit of GABAᴀRs that Promotes Inhibitory Synaptogenesis and Resilience to Seizures

The fidelity of inhibitory neurotransmission is dependent on the accumulation of γ-aminobutyric acid type A receptors (GABAARs) at the appropriate synaptic sites. Synaptic GABAARs are constructed from α(1-3), β(1-3), and γ2 subunits, and neurons can target these subtypes to specific synapses. Here, we identify a 15-amino acid inhibitory synapse targeting motif (ISTM) within the α2 subunit that promotes the association between GABAARs and the inhibitory scaffold proteins collybistin and gephyrin. Using mice in which the ISTM has been introduced into the α1 subunit (Gabra1-2 mice), we show that the ISTM is critical for axo-axonic synapse formation, the efficacy of GABAergic neurotransmission, and seizure sensitivity. The Gabra1-2 mutation rescues seizure-induced lethality in Gabra2-1 mice, which lack axo-axonic synapses due to the deletion of the ISTM from the α2 subunit. Taken together, our data demonstrate that the ISTM plays a critical role in promoting inhibitory synapse formation, both in the axonic and somatodendritic compartments

Slugging their way to immortality: driving mammary epithelial cells into a stem cell-like state

Delineating the molecular factors that define and maintain the mammary stem cell state is vital for understanding normal development and tumourigenesis. A recent study by Guo and colleagues identifies two master transcriptional regulators of mammary stem cells, Slug and Sox9, ectopic expression of which confers stem cell attributes on differentiated mammary epithelial cells. Slug and Sox9 expression was also shown to determine in vivo metastatic potential of human breast cancer cell lines. Understanding these factors in the context of normal lineage differentiation is an important step toward elucidating the mammary epithelial cell hierarchy and the origins of cancer stem cells

Nonsteroidal anti-inflammatory drugs (NSAIDs), cyxlooxygenase-2 selective inhibitors (coxibs) and gastrointestinal harm: review of clinical trials and clinical practice

BACKGROUND: Gastrointestinal harm, known to occur with NSAIDs, is thought to be lower with NSAID and gastroprotective agent, and with inhibitors selective to cyclooxygenase-2 (coxibs) at usual plasma concentrations. We examine competing strategies for available evidence of reduced gastrointestinal bleeding in clinical trials and combine this evidence with evidence from clinical practice on whether the strategies work in the real world, whether guidance on appropriate prescribing is followed, and whether patients adhere to the strategies. METHODS: We used a series of systematic literature searches to find full publications of relevant studies for evidence about the efficacy of these different gastroprotection strategies in clinical trials, and for evidence that they worked and were adhered to in clinical practice – whether they were effective. We chose to use good quality systematic reviews and meta-analyses when they were available. RESULTS: Evidence of efficacy of coxibs compared to NSAIDs for upper gastrointestinal bleeding was strong, with consistent reductions in events of about 50% in large randomised trials (34,460 patients), meta-analyses of randomised trials (52,474 patients), and large observational studies in clinical practice (3,093 bleeding events). Evidence on the efficacy of NSAID plus gastroprotection with acid suppressants (proton pump inhibitors, PPIs, and histamine antagonists, H2As) was based mainly on the surrogate measure of endoscopic ulcers. The limited information on damage to the bowel suggested that NSAID plus PPI was more damaging than coxibs. Eleven observational studies studied 1.6 million patients, of whom 911,000 were NSAID users, and showed that 76% (range 65% to 90%) of patients with at least one gastrointestinal risk factor received no prescription for gastroprotective agent with an NSAID. The exception was a cohort of US veterans with previous gastrointestinal bleeding, where 75% had gastroprotection with an NSAID. When gastroprotection was prescribed, it was often described as inadequate. A single study suggested that patient adherence to prescribed gastroprotection was low. CONCLUSION: Evidence for efficacy of gastroprotection strategies with NSAIDs is limited. In clinical practice few patients who need gastroprotection get it, and those who get it may not take it. For coxibs, gastroprotection is inherent, although probably not complete

Surrogate markers and survival in women receiving first-line combination anthracycline chemotherapy for advanced breast cancer

Surrogate markers may help predict the effects of first-line treatment on survival. This metaregression analysis examines the relationship between several surrogate markers and survival in women with advanced breast cancer after receiving first-line combination anthracycline chemotherapy 5-fluorouracil, adriamycin and cyclophosphamide (FAC) or 5-fluorouracil, epirubicin and cyclophosphamide (FEC) . From a systematic literature review, we identified 42 randomised trials. The surrogate markers were complete or partial tumour response, progressive disease and time to progression. The treatment effect on survival was quantified by the hazard ratio. The treatment effect on each surrogate marker was quantified by the odds ratio (or ratio of median time to progression). The relationship between survival and each surrogate marker was assessed by a weighted linear regression of the hazard ratio against the odds ratio. There was a significant linear association between survival and complete or partial tumour response (P<0.001, R2=34%), complete tumour response (P=0.02, R2=12%), progressive disease (P<0.001, R2=38%) and time to progression (P<0.0001, R2=56%); R2 is the proportion of the variability in the treatment effect on survival that is explained by the treatment effect on the surrogate marker. Time to progression may be a useful surrogate marker for predicting survival in women receiving first-line anthracycline chemotherapy and could be used to estimate the survival benefit in future trials of first-line chemotherapy compared to FAC or FEC. The other markers, tumour response and progressive disease, were less good