Detection of autism spectrum disorder-related pathogenic trio variants by a novel structure-based approach.

BACKGROUND: Glutamatergic synapse dysfunction is believed to underlie the development of Autism Spectrum Disorder (ASD) and Intellectual Disability (ID) in many individuals. However, identification of genetic markers that contribute to synaptic dysfunction in these individuals is notoriously difficult. Based on genomic analysis, structural modeling, and functional data, we recently established the involvement of the TRIO-RAC1 pathway in ASD and ID. Furthermore, we identified a pathological de novo missense mutation hotspot in TRIO's GEF1 domain. ASD/ID-related missense mutations within this domain compromise glutamatergic synapse function and likely contribute to the development of ASD/ID. The number of ASD/ID cases with mutations identified within TRIO's GEF1 domain is increasing. However, tools for accurately predicting whether such mutations are detrimental to protein function are lacking. METHODS: Here we deployed advanced protein structural modeling techniques to predict potential de novo pathogenic and benign mutations within TRIO's GEF1 domain. Mutant TRIO-9 constructs were generated and expressed in CA1 pyramidal neurons of organotypic cultured hippocampal slices. AMPA receptor-mediated postsynaptic currents were examined in these neurons using dual whole-cell patch clamp electrophysiology. We also validated these findings using orthogonal co-immunoprecipitation and fluorescence lifetime imaging (FLIM-FRET) experiments to assay TRIO mutant overexpression effects on TRIO-RAC1 binding and on RAC1 activity in HEK293/T cells. RESULTS: Missense mutations in TRIO's GEF1 domain that were predicted to disrupt TRIO-RAC1 binding or stability were tested experimentally and found to greatly impair TRIO-9's influence on glutamatergic synapse function. In contrast, missense mutations in TRIO's GEF1 domain that were predicted to have minimal effect on TRIO-RAC1 binding or stability did not impair TRIO-9's influence on glutamatergic synapse function in our experimental assays. In orthogonal assays, we find most of the mutations predicted to disrupt binding display loss of function but mutants predicted to disrupt stability do not reflect our results from neuronal electrophysiological data. LIMITATIONS: We present a method to predict missense mutations in TRIO's GEF1 domain that may compromise TRIO function and test for effects in a limited number of assays. Possible limitations arising from the model systems employed here can be addressed in future studies. Our method does not provide evidence for whether these mutations confer ASD/ID risk or the likelihood that such mutations will result in the development of ASD/ID. CONCLUSIONS: Here we show that a combination of structure-based computational predictions and experimental validation can be employed to reliably predict whether missense mutations in the human TRIO gene impede TRIO protein function and compromise TRIO's role in glutamatergic synapse regulation. With the growing accessibility of genome sequencing, the use of such tools in the accurate identification of pathological mutations will be instrumental in diagnostics of ASD/ID.

Human BDNF/TrkB variants impair hippocampal synaptogenesis and associate with neurobehavioural abnormalities.

Brain-derived neurotrophic factor (BDNF) signals through its high affinity receptor Tropomyosin receptor kinase-B (TrkB) to regulate neuronal development, synapse formation and plasticity. In rodents, genetic disruption of Bdnf and TrkB leads to weight gain and a spectrum of neurobehavioural phenotypes. Here, we functionally characterised a de novo missense variant in BDNF and seven rare variants in TrkB identified in a large cohort of people with severe, childhood-onset obesity. In cells, the E183K BDNF variant resulted in impaired processing and secretion of the mature peptide. Multiple variants in the kinase domain and one variant in the extracellular domain of TrkB led to a loss of function through multiple signalling pathways, impaired neurite outgrowth and dominantly inhibited glutamatergic synaptogenesis in hippocampal neurons. BDNF/TrkB variant carriers exhibited learning difficulties, impaired memory, hyperactivity, stereotyped and sometimes, maladaptive behaviours. In conclusion, human loss of function BDNF/TrkB variants that impair hippocampal synaptogenesis may contribute to a spectrum of neurobehavioural disorders.

Regulatory light chain phosphorylation augments length-dependent contraction in PTU-treated rats.

Force production by actin-myosin cross-bridges in cardiac muscle is regulated by thin-filament proteins and sarcomere length (SL) throughout the heartbeat. Prior work has shown that myosin regulatory light chain (RLC), which binds to the neck of myosin heavy chain, increases cardiac contractility when phosphorylated. We recently showed that cross-bridge kinetics slow with increasing SLs, and that RLC phosphorylation amplifies this effect, using skinned rat myocardial strips predominantly composed of the faster α-cardiac myosin heavy chain isoform. In the present study, to assess how RLC phosphorylation influences length-dependent myosin function as myosin motor speed varies, we used a propylthiouracil (PTU) diet to induce >95% expression of the slower ß-myosin heavy chain isoform in rat cardiac ventricles. We measured the effect of RLC phosphorylation on Ca2+-activated isometric contraction and myosin cross-bridge kinetics (via stochastic length perturbation analysis) in skinned rat papillary muscle strips at 1.9- and 2.2-µm SL. Maximum tension and Ca2+ sensitivity increased with SL, and RLC phosphorylation augmented this response at 2.2-µm SL. Subtle increases in viscoelastic myocardial stiffness occurred with RLC phosphorylation at 2.2-µm SL, but not at 1.9-µm SL, thereby suggesting that RLC phosphorylation increases ß-myosin heavy chain binding or stiffness at longer SLs. The cross-bridge detachment rate slowed as SL increased, providing a potential mechanism for prolonged cross-bridge attachment to augment length-dependent activation of contraction at longer SLs. Length-dependent slowing of ß-myosin heavy chain detachment rate was not affected by RLC phosphorylation. Together with our previous studies, these data suggest that both α- and ß-myosin heavy chain isoforms show a length-dependent activation response and prolonged myosin attachment as SL increases in rat myocardial strips, and that RLC phosphorylation augments length-dependent activation at longer SLs. In comparing cardiac isoforms, however, we found that ß-myosin heavy chain consistently showed greater length-dependent sensitivity than α-myosin heavy chain. Our work suggests that RLC phosphorylation is a vital contributor to the regulation of myocardial contractility in both cardiac myosin heavy chain isoforms.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis.

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 × 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Two Na+ Sites Control Conformational Change in a Neurotransmitter Transporter Homolog.

In LeuT, a prokaryotic homolog of neurotransmitter transporters, Na(+) stabilizes outward-open conformational states. We examined how each of the two LeuT Na(+) binding sites contributes to Na(+)-dependent closure of the cytoplasmic pathway using biochemical and biophysical assays of conformation. Mutating either of two residues that contribute to the Na2 site completely prevented cytoplasmic closure in response to Na(+), suggesting that Na2 is essential for this conformational change, whereas Na1 mutants retained Na(+) responsiveness. However, mutation of Na1 residues also influenced the Na(+)-dependent conformational change in ways that varied depending on the position mutated. Computational analyses suggest those mutants influence the ability of Na1 binding to hydrate the substrate pathway and perturb an interaction network leading to the extracellular gate. Overall, the results demonstrate that occupation of Na2 stabilizes outward-facing conformations presumably through a direct interaction between Na(+) and transmembrane helices 1 and 8, whereas Na(+) binding at Na1 influences conformational change through a network of intermediary interactions. The results also provide evidence that N-terminal release and helix motions represent distinct steps in cytoplasmic pathway opening.

Physico-chemical requirements and kinetics of membrane fusion of flavivirus-like particles.

Flaviviruses deliver their RNA genome into the host-cell cytoplasm by fusing their lipid envelope with a cellular membrane. Expression of the flavivirus pre-membrane and envelope glycoprotein genes in the absence of other viral genes results in the spontaneous assembly and secretion of virus-like particles (VLPs) with membrane fusion activity. Here, we examined the physico-chemical requirements for membrane fusion of VLPs from West Nile and Japanese encephalitis viruses. In a bulk fusion assay, optimal hemifusion (or lipid mixing) efficiencies were observed at 37 °C. Fusion efficiency increased with decreasing pH; half-maximal hemifusion was attained at pH 5.6. The anionic lipids bis(monoacylglycero)phosphate and phosphatidylinositol-3-phosphate, when present in the target membrane, significantly enhanced fusion efficiency, consistent with the emerging model that flaviviruses fuse with intermediate-to-late endosomal compartments, where these lipids are most abundant. In a single-particle fusion assay, VLPs catalysed membrane hemifusion, tracked as lipid mixing with the cellular membrane, on a timescale of 7-20 s after acidification. Lipid mixing kinetics suggest that hemifusion is a kinetically complex, multistep process.

Core binding factor beta plays a critical role by facilitating the assembly of the Vif-cullin 5 E3 ubiquitin ligase.

UNLABELLED: The HIV-1 virion infectivity factor (Vif) targets the cellular cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) for degradation via the host ubiquitin-proteasome pathway. Vif recruits a cellular E3 ubiquitin ligase to polyubiquitinate A3G/F. The activity of Vif critically depends on the cellular core binding factor beta (CBFß). In this study, we investigated the Vif-CBFß interaction and the role of CBFß in the E3 ligase assembly. Vif-CBFß interaction requires an extensive region of Vif spanning most of its amino terminus and zinc finger region, and cullin 5 (Cul5) binding enhances the stability of the Vif-CBFß interaction. Our results further demonstrate that CBFß plays a critical role in facilitating Cul5 binding to the Vif/elongin B/elongin C complex. Vif, with or without bound substrate, is unable to bind Cul5 in the absence of CBFß. These studies support the notion that CBFß serves as a molecular chaperone to facilitate Vif-E3 ligase assembly. IMPORTANCE: The host antiviral restriction factors A3G/F inhibit viral replication. The HIV-1 protein Vif targets A3G/F for degradation. This immune evasion activity of Vif is dependent on the cellular factor CBFß. Multiple regions of Vif are known to be important for Vif function, but the mechanisms are unclear. The studies described here provide important information about the Vif-CBFß interaction interface and the function of CBFß in E3 ligase assembly. In particular, our comprehensive Vif-CBFß interface mapping results help to delineate the role of various Vif regions, determining if they are important for binding CBFß or A3G/F. Furthermore, our studies reveal an important potential mechanism of CBFß that has not been shown before. Our results suggest that CBFß may serve as a molecular chaperone to enable Vif to adopt an appropriate conformation for interaction with the Cul5-based E3 ligase. This study advances our understanding of how CBFß facilitates the Vif-mediated degradation of APOBEC3 proteins.

α-Synuclein can inhibit SNARE-mediated vesicle fusion through direct interactions with lipid bilayers.

The native function of α-synuclein is thought to involve regulation of synaptic vesicle trafficking. Recent work has also implicated a role in neurotransmission, possibly through interactions with the proteins involved in synaptic vesicle fusion. Here, we demonstrate that α-synuclein inhibits SNARE-mediated vesicle fusion through binding the membrane, without a direct interaction between α-synuclein and any of the SNARE proteins. This work supports a model in which α-synuclein plays a role in the regulation of vesicle fusion by modulating properties of the lipid bilayer.

The conformational ensembles of α-synuclein and tau: combining single-molecule FRET and simulations.

Intrinsically disordered proteins (IDPs) are increasingly recognized for their important roles in a range of biological contexts, both in normal physiological function and in a variety of devastating human diseases. However, their structural characterization by traditional biophysical methods, for the purposes of understanding their function and dysfunction, has proved challenging. Here, we investigate the model IDPs α-Synuclein (αS) and tau, that are involved in major neurodegenerative conditions including Parkinson's and Alzheimer's diseases, using excluded volume Monte Carlo simulations constrained by pairwise distance distributions from single-molecule fluorescence measurements. Using this, to our knowledge, novel approach we find that a relatively small number of intermolecular distance constraints are sufficient to accurately determine the dimensions and polymer conformational statistics of αS and tau in solution. Moreover, this method can detect local changes in αS and tau conformations that correlate with enhanced aggregation. Constrained Monte Carlo simulations produce ensembles that are in excellent agreement both with experimental measurements on αS and tau and with all-atom, explicit solvent molecular dynamics simulations of αS, with much lower configurational sampling requirements and computational expense.