Crosstalk between ion channels and small GTPases is critical during homeostasis and disease, but little is known about the structural underpinnings of these interactions. TRPV4 is a polymodal, ...calcium-permeable cation channel that has emerged as a potential therapeutic target in multiple conditions. Gain-of-function mutations also cause hereditary neuromuscular disease. Here, we present cryo-EM structures of human TRPV4 in complex with RhoA in the ligand-free, antagonist-bound closed, and agonist-bound open states. These structures reveal the mechanism of ligand-dependent TRPV4 gating. Channel activation is associated with rigid-body rotation of the intracellular ankyrin repeat domain, but state-dependent interaction with membrane-anchored RhoA constrains this movement. Notably, many residues at the TRPV4-RhoA interface are mutated in disease and perturbing this interface by introducing mutations into either TRPV4 or RhoA increases TRPV4 channel activity. Together, these results suggest that RhoA serves as an auxiliary subunit for TRPV4, regulating TRPV4-mediated calcium homeostasis and disruption of TRPV4-RhoA interactions can lead to TRPV4-related neuromuscular disease. These insights will help facilitate TRPV4 therapeutics development.
Several recent studies link parental environments to phenotypes in subsequent generations. In this work, we investigate the mechanism by which paternal diet affects offspring metabolism. Protein ...restriction in mice affects small RNA (sRNA) levels in mature sperm, with decreased let-7 levels and increased amounts of 5’ fragments of glycine transfer RNAs (tRNAs). In testicular sperm, tRNA fragments are scarce but increase in abundance as sperm mature in the epididymis. Epididymosomes (vesicles that fuse with sperm during epididymal transit) carry RNA payloads matching those of mature sperm and can deliver RNAs to immature sperm in vitro. Functionally, tRNA-glycine-GCC fragments repress genes associated with the endogenous retroelement MERVL, in both embryonic stem cells and embryos. Our results shed light on sRNA biogenesis and its dietary regulation during posttesticular sperm maturation, and they also link tRNA fragments to regulation of endogenous retroelements active in the preimplantation embryo.
Incomplete reporting of study methods and results has become a focal point for failures in the reproducibility and translation of findings from preclinical research. Here we demonstrate that ...incomplete reporting of preclinical research is not limited to a few elements of research design, but rather is a broader problem that extends to the reporting of the methods and results. We evaluated 47 preclinical research studies from a systematic review of acute lung injury that use mesenchymal stem cells (MSCs) as a treatment. We operationalized the ARRIVE (Animal Research: Reporting of In Vivo Experiments) reporting guidelines for pre-clinical studies into 109 discrete reporting sub-items and extracted 5,123 data elements. Overall, studies reported less than half (47%) of all sub-items (median 51 items; range 37-64). Across all studies, the Methods Section reported less than half (45%) and the Results Section reported less than a third (29%). There was no association between journal impact factor and completeness of reporting, which suggests that incomplete reporting of preclinical research occurs across all journals regardless of their perceived prestige. Incomplete reporting of methods and results will impede attempts to replicate research findings and maximize the value of preclinical studies.
The Iowa Gambling Task (IGT) is used to assess decision-making in clinical populations. The original IGT does not disambiguate reward and punishment learning; however, an adaptation of the task, the ...“play-or-pass” IGT, was developed to better distinguish between reward and punishment learning. We evaluated the test-retest reliability of measures of reward and punishment learning from the play-or-pass IGT and examined associations with self-reported measures of reward/punishment sensitivity and internalizing symptoms. Participants completed the task across two sessions, and we calculated mean-level differences and rank-order stability of behavioral measures across the two sessions using traditional scoring, involving session-wide choice proportions, and computational modeling, involving estimates of different aspects of trial-level learning. Measures using both approaches were reliable; however, computational modeling provided more insights regarding between-session changes in performance, and how performance related to self-reported measures of reward/punishment sensitivity and internalizing symptoms. Our results show promise in using the play-or-pass IGT to assess decision-making; however, further work is still necessary to validate the play-or-pass IGT.
Abstract
The cation channel transient receptor potential vanilloid 4 (TRPV4) is one of the few identified ion channels that can directly cause inherited neurodegeneration syndromes, but the molecular ...mechanisms are unknown. Here, we show that in vivo expression of a neuropathy-causing TRPV4 mutant (TRPV4
R269C
) causes dose-dependent neuronal dysfunction and axonal degeneration, which are rescued by genetic or pharmacological blockade of TRPV4 channel activity. TRPV4
R269C
triggers increased intracellular Ca
2+
through a Ca
2+
/calmodulin-dependent protein kinase II (CaMKII)-mediated mechanism, and CaMKII inhibition prevents both increased intracellular Ca
2+
and neurotoxicity in
Drosophila
and cultured primary mouse neurons. Importantly, TRPV4 activity impairs axonal mitochondrial transport, and TRPV4-mediated neurotoxicity is modulated by the Ca
2+
-binding mitochondrial GTPase Miro. Our data highlight an integral role for CaMKII in neuronal TRPV4-associated Ca
2+
responses, the importance of tightly regulated Ca
2+
dynamics for mitochondrial axonal transport, and the therapeutic promise of TRPV4 antagonists for patients with TRPV4-related neurodegenerative diseases.
Presynaptic resting Ca2+ influences synaptic vesicle (SV) release probability. Here, we report that a TRPV channel, Inactive (Iav), maintains presynaptic resting Ca2+ by promoting Ca2+ release from ...the endoplasmic reticulum in Drosophila motor neurons, and is required for both synapse development and neurotransmission. We find that Iav activates the Ca2+/calmodulin-dependent protein phosphatase calcineurin, which is essential for presynaptic microtubule stabilization at the neuromuscular junction. Thus, loss of Iav induces destabilization of presynaptic microtubules, resulting in diminished synaptic growth. Interestingly, expression of human TRPV1 in Iav-deficient motor neurons rescues these defects. We also show that the absence of Iav causes lower SV release probability and diminished synaptic transmission, whereas Iav overexpression elevates these synaptic parameters. Together, our findings indicate that Iav acts as a key regulator of synaptic development and function by influencing presynaptic resting Ca2+.
•Iav-dependent ER Ca2+ release promotes NMJ synapse development by regulating the activity of calcineurin, which dephosphorylates presynaptic microtubule-associated proteins•Expression of human TRPV1, but not TRPV4, rescues the synaptic growth defects observed in the iav mutants•Iav plays a dose-dependent role in maintaining presynaptic resting Ca2+ levels and synaptic transmission
Wong et al. demonstrate that the Drosophila TRPV channel, Inactive (Iav), functions cell autonomously in motor neurons to promote Ca2+ release from the endoplasmic reticulum (ER), which is required for neuromuscular junction (NMJ) synapse development and function.
One of the greatest challenges in healthcare is how to best translate research evidence into clinical practice, which includes how to change health-care professionals' behaviours. A commonly held ...view is that multifaceted interventions are more effective than single-component interventions. The purpose of this study was to conduct an overview of systematic reviews to evaluate the effectiveness of multifaceted interventions in comparison to single-component interventions in changing health-care professionals' behaviour in clinical settings.
The Rx for Change database, which consists of quality-appraised systematic reviews of interventions to change health-care professional behaviour, was used to identify systematic reviews for the overview. Dual, independent screening and data extraction was conducted. Included reviews used three different approaches (of varying methodological robustness) to evaluate the effectiveness of multifaceted interventions: (1) effect size/dose-response statistical analyses, (2) direct (non-statistical) comparisons of multifaceted to single interventions and (3) indirect comparisons of multifaceted to single interventions.
Twenty-five reviews were included in the overview. Three reviews provided effect size/dose-response statistical analyses of the effectiveness of multifaceted interventions; no statistical evidence of a relationship between the number of intervention components and the effect size was found. Eight reviews reported direct (non-statistical) comparisons of multifaceted to single-component interventions; four of these reviews found multifaceted interventions to be generally effective compared to single interventions, while the remaining four reviews found that multifaceted interventions had either mixed effects or were generally ineffective compared to single interventions. Twenty-three reviews indirectly compared the effectiveness of multifaceted to single interventions; nine of which also reported either a statistical (dose-response) analysis (N = 2) or a non-statistical direct comparison (N = 7). The majority (N = 15) of reviews reporting indirect comparisons of multifaceted to single interventions showed similar effectiveness for multifaceted and single interventions when compared to controls. Of the remaining eight reviews, six found single interventions to be generally effective while multifaceted had mixed effectiveness.
This overview of systematic reviews offers no compelling evidence that multifaceted interventions are more effective than single-component interventions.
TRPV4 is a cell surface-expressed calcium-permeable cation channel that mediates cell-specific effects on cellular morphology and function. Dominant missense mutations of TRPV4 cause distinct, ...tissue-specific diseases, but the pathogenic mechanisms are unknown. Mutations causing peripheral neuropathy localize to the intracellular N-terminal domain whereas skeletal dysplasia mutations are in multiple domains. Using an unbiased screen, we identified the cytoskeletal remodeling GTPase RhoA as a TRPV4 interactor. TRPV4-RhoA binding occurs via the TRPV4 N-terminal domain, resulting in suppression of TRPV4 channel activity, inhibition of RhoA activation, and extension of neurites in vitro. Neuropathy but not skeletal dysplasia mutations disrupt TRPV4-RhoA binding and cytoskeletal outgrowth. However, inhibition of RhoA restores neurite length in vitro and in a fly model of TRPV4 neuropathy. Together these results identify RhoA as a critical mediator of TRPV4-induced cell structure changes and suggest that disruption of TRPV4-RhoA binding may contribute to tissue-specific toxicity of TRPV4 neuropathy mutations.
Mycobacterium tuberculosis (Mtb) infection is notoriously difficult to treat. Treatment efficacy is limited by Mtb's intrinsic drug resistance, as well as its ability to evolve acquired resistance to ...all antituberculars in clinical use. A deeper understanding of the bacterial pathways that influence drug efficacy could facilitate the development of more effective therapies, identify new mechanisms of acquired resistance, and reveal overlooked therapeutic opportunities. Here we developed a CRISPR interference chemical-genetics platform to titrate the expression of Mtb genes and quantify bacterial fitness in the presence of different drugs. We discovered diverse mechanisms of intrinsic drug resistance, unveiling hundreds of potential targets for synergistic drug combinations. Combining chemical genetics with comparative genomics of Mtb clinical isolates, we further identified several previously unknown mechanisms of acquired drug resistance, one of which is associated with a multidrug-resistant tuberculosis outbreak in South America. Lastly, we found that the intrinsic resistance factor whiB7 was inactivated in an entire Mtb sublineage endemic to Southeast Asia, presenting an opportunity to potentially repurpose the macrolide antibiotic clarithromycin to treat tuberculosis. This chemical-genetic map provides a rich resource to understand drug efficacy in Mtb and guide future tuberculosis drug development and treatment.
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