Biology subjects

Wood, M.

Publications and source records attributed to Wood, M..

28 records · Page 2Linked to original sources

bioRxiv2021

Dysregulation of the Tweak/Fn14 pathway in skeletal muscle of spinal muscular atrophy mice

Spinal muscular atrophy (SMA) is a childhood neuromuscular disorder caused by depletion of the survival motor neuron (SMN) protein. SMA is characterized by the selective death of spinal cord motor neurons, leading to progressive muscle wasting. Loss of skeletal muscle in SMA is a combination of denervation-induced muscle atrophy and intrinsic muscle pathologies. Elucidation of the pathways involved is essential to identify the key molecules that contribute to and sustain muscle pathology. The tumor necrosis factor-like weak inducer of apoptosis (TWEAK)/TNF receptor superfamily member fibroblast growth factor inducible 14 (Fn14) pathway has been shown to play a critical role in the regulation of denervation-induced muscle atrophy as well as muscle proliferation, differentiation and metabolism in adults. However, it is not clear whether this pathway would be important in highly dynamic and developing muscle. We thus investigated the potential role of the TWEAK/Fn14 pathway in SMA muscle pathology, using the severe Taiwanese Smn-/-;SMN2 and the less severe Smn2B/- SMA mice, which undergo a progressive neuromuscular decline in the first three post-natal weeks. Here, we report significantly dysregulated expression of the TWEAK/Fn14 pathway during disease progression in skeletal muscle of the two SMA mouse models. In addition, siRNA-mediated Smn knockdown in C2C12 myoblasts suggests a genetic interaction between Smn and the TWEAK/Fn14 pathway. Further analyses of SMA, Tweak-/- and Fn14-/- mice revealed dysregulated myopathy, myogenesis and glucose metabolism pathways as a common skeletal muscle feature, and providing further evidence in support of a relationship between the TWEAK/Fn14 pathway and Smn. Finally, a pharmacological intervention (Fc-TWEAK) to upregulate the activity of the TWEAK/Fn14 pathway improved disease phenotypes in the two SMA mouse models. Our study provides novel mechanistic insights into the molecular players that contribute to muscle pathology in SMA and into the role of the TWEAK/Fn14 pathway in developing muscle.

pathology↗

bioRxiv2021

A Gene Replacement Humanization Platform for Rapid Functional Testing of Clinical Variants in Epilepsy-associated STXBP1.

PurposeFunctional evidence is a pillar of variant interpretation according to ACMG guidelines. Functional evidence can be obtained in a variety of models and assay systems, including patient-derived tissues and iPSCs, in vitro cellular assays, and in vivo assays. Here we evaluate the reliability and practicality of variant interpretation in the small animal model, C. elegans, through a series of experiments evaluating the function of syntaxin binding protein, STXBP1, a well-known causative gene for Early infantile epileptic encephalopathy 1 (EIEE1). MethodsUsing CRISPR, we replaced the coding sequence for unc-18 with the coding sequence for the human ortholog STXBP1. Next, we used CRISPR to introduce precise point mutations in the human STXBP1 coding sequence, reflecting three clinical categories (benign, pathogenic, and variants of uncertain significance (VUS)). We quantified 26 features of the resulting worms movement to train Random Forest (RF) and Support Vector Machines (SVM) machine learning classifiers on known pathogenic and benign variants. We characterized the classifiers, and then used the behavioral data from the VUS-expressing animals to predict the categorization of the VUS. ResultsWhereas knock-out worms without unc-18 are severely impaired in motor function, worms expressing STXBP1 in its place have restored motor function. We produced worms with STXBP1 variants previously classified by ACMG criteria, including 25 benign variants, 32 pathogenic, and 24 variants of uncertain significance (VUS). Using either SVM or RF classifiers, we were able to obtain a sensitivity of 0.84-0.97 on known benign and pathogenic strains. By comparing multiple ML classification methods, we were able to classify 9 of the VUS as functionally abnormal, suggesting that these VUS are likely to be pathogenic. ConclusionsWe demonstrate that automated analysis of a small animal system is an effective, scalable, and fast way to understand functional consequences of variants in STXBP1, one of the most common causes of genetic epilepsies and neurodevelopmental disorders.

genetics↗

bioRxiv2021

Systemic antibody-oligonucleotide delivery to the central nervous system ameliorates mouse models of spinal muscular atrophy

Antisense oligonucleotides (ASOs) have emerged as one of the most innovative new genetic drug modalities, however, the high molecular weight limits their bioavailability for otherwise treatable neurological disorders. We investigated conjugation of ASOs to an antibody against the murine transferrin receptor (TfR), 8D3130, and evaluated it via systemic administration in mouse models of the neurodegenerative disease, spinal muscular atrophy (SMA). SMA like several other neurological and neuromuscular diseases, is treatable with single-stranded ASOs, inducing splice modulation of the survival motor neuron 2 (SMN2) gene. Administration of 8D3130-ASO conjugate resulted in bioavailability of 2.7% of the injected dose in brain. Additionally, 8D3130-ASO yielded therapeutically high levels of SMN2 splicing in the central nervous system of mildly affected adult SMA mice and resulted in extended survival of severe SMA mice. Systemic delivery of nucleic acid therapies with brain targeting antibodies offers powerful translational potential for future treatments of neuromuscular and neurodegenerative diseases.

molecular biology↗

bioRxiv2021

Dystrophin regulates peripheral circadian SRF signalling

Dystrophin is a sarcolemmal protein essential for muscle contraction and maintenance, absence of which leads to the devastating muscle wasting disease Duchenne muscular dystrophy (DMD)[1, 2]. Dystrophin has an actin-binding domain [3-5], which specifically binds and stabilises filamentous (F)-actin[6], an integral component of the RhoA-actin-serum response factor (SRF)-pathway[7]. The RhoA-actin-SRF-pathway plays an essential role in circadian signalling whereby the hypothalamic suprachiasmatic nucleus, transmits systemic cues to peripheral tissues, activating SRF and transcription of clock target genes[8, 9]. Given dystrophin binds F-actin and disturbed SRF-signalling disrupts clock entrainment, we hypothesised that dystrophin loss causes circadian deficits. Here we show for the first time alterations in the RhoA-actin-SRF-signalling-pathway, in both dystrophin-deficient myotubes and dystrophic mouse models. Specifically, we demonstrate reduced F/G-actin ratios and nuclear MRTF, dysregulation of core clock and downstream target-genes, and down-regulation of key circadian genes in muscle biopsies from DMD patients harbouring an array of mutations. Further, disrupted circadian locomotor behaviour was observed in dystrophic mice indicative of disrupted SCN signalling, and indeed dystrophin protein was absent in the SCN of dystrophic animals. Dystrophin is thus a critically important component of the RhoA-actin-SRF-pathway and a novel mediator of circadian signalling in peripheral tissues, loss of which leads to circadian dysregulation.

cell biology↗

bioRxiv2021

Pepsickle rapidly and accurately predicts proteasomal cleavage sites for improved neoantigen identification

Proteasomal cleavage is a key component in protein turnover, as well as antigen presentation and subsequent immune response. Herein we present pepsickle, an open-source tool for proteasomal cleavage prediction with better in vivo prediction performance (AUC) and computational speed than current models available in the field, and with the ability to predict sites based on both constitutive and immunoproteasome profiles. Post-hoc filtering of predicted patient neoepitopes using pepsickle significantly enriches for immune-responsive epitopes and may represent a significant opportunity to improve current epitope prediction and vaccine development pipelines.

bioinformatics↗

bioRxiv2021

RASSF1C oncogene elicits amoeboid invasion, cancer stemness and invasive EVs via a novel SRC/Rho axis

Cell plasticity is a crucial hallmark leading to cancer metastasis. Upregulation of Rho/ROCK pathway drives actomyosin contractility, protrusive forces and contributes to the occurrence of highly invasive amoeboid cells in tumors. Cancer stem cells are similarly associated with metastasis, but how these populations arise in tumors is not fully understood. Here we show that the novel oncogene RASSF1C drives mesenchymal to amoeboid transition and stem cell attributes in breast cancer cells. Mechanistically, RASSF1C activates Rho/ROCK via SRC mediated RhoGDI inhibition, resulting in generation of actomyosin contractility. Moreover, we demonstrate that amoeboid cells display the cancer stem cell markers CD133, ALDH1 and the pluripotent marker Nanog; are accompanied by higher invasive potential in vitro and in vivo; and employ extracellular vesicles to transfer the invasive phenotype to target cells and tissue. Importantly, the underlying RASSF1C driven biological processes concur to explain clinical data: namely, methylation of the RASSF1C promoter correlates with better survival in early stage breast cancer patients. Therefore, we propose the use of RASSF1 gene promoter methylation status as a biomarker for patient stratification.

cancer biology↗

bioRxiv2020

GAI MoRFs Regulate Cleft and Channel Binding Pathways for Gibberellin in GID1A

AbstractThe hormone gibberellin (GA) promotes arabidopsis growth by enhancing binding between GA Insensitive DELLA transcriptional repressors and GA Insensitive Dwarf 1 (GID1) receptors to regulate DELLA degradation. The binding mechanism for GA was elucidated by employing a computational study of dissociations of the N-terminus of the DELLA family member GAI (GA Insensitive transcriptional repressor) from the GID1A receptor in the presence and absence of bound GA, and of GA from GID1A in the presence and absence of GAI. The tRAMD method was employed to deduce egression pathways for a diverse set of GA molecules (GA(x)). Two pathways in the form of a newly identified cleft and a previously identified channel are prevalent. The cleft pathway is open in the absence of GAI. Upon GAI binding, the cleft route is blocked, resulting in a slower process for GA(x) to exit and enter the binding pocket through the channel. Several binding pocket residues are identified as gate-keepers to the channel. Molecular recognition features (MoRFs) found in the disordered signaling protein GAI affect GA(x) binding and GID1A dynamics. A three-step synergistic binding cycle is proposed where GAI MoRFs regulate the process. Rapid binding takes place through the cleft where little to no distinctions are made between major and less active forms of GA(x). After GAI is bound to the GA(x) {middle dot} GID1A complex, the channel supports a rectification process that increases the retention of major active forms of GA within the binding pocket. Both the cleft and channel contact residues to GA(x) are markedly conserved in a GID1 phylogeny, suggesting this binding process in the GID1 {middle dot} DELLA GA-receptor complex represents a general paradigm for GA binding. Non-specific GA binding assists binding of GAI, which then helps to select the major active forms of the hormone and induce a downstream signalling cascade in response to bioactive GA. Non-expert Summary StatementGibberellins are plant hormones essential for growth and development. The DELLA proteins are a disordered family of repressors that transcriptionally repress GA responsive genes. Degradation of DELLA proteins in response to GA results in GA-responsive genes being upregulated. Binding of GA to the GA-Insensitive Dwarf 1 receptor (GID1) facilitates binding of DELLA to the GA {middle dot} GID1 complex. Through computational modelling and phylogenetic analyses, we identified a new GA binding cleft that is blocked by DELLA binding and a three-step mechanism for the GA {middle dot} DELLA {middle dot} GID1 complex that also involves the known GA binding channel. We propose a dual (cleft/channel) pathway that allows access to the binding pocket as a paradigm for selection of specific GA forms among a mixture of major active and inactive forms. The cleft is less selective, but preference for active GA in the binding pocket of GID1A is amplified by expunging inactive GA forms, followed by recruiting active forms through the more selective channel. This mechanism allows plants to sense concentration changes of GA with high specificity to enable certain GA variants to trigger specific signalling events. These novel insights into the receptor mechanism in part may explain the large number of different GA forms that exist in nature. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/422840v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@a7b6e5org.highwire.dtl.DTLVardef@d89799org.highwire.dtl.DTLVardef@1c8ee74org.highwire.dtl.DTLVardef@9f31c6_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

Engineered extracellular vesicle decoy receptor-mediated modulation of the IL6 trans-signalling pathway in muscle

The cytokine interleukin 6 (IL6) is a key mediator of inflammation that contributes to skeletal muscle pathophysiology. IL6 activates target cells by two different mechanisms, the classical and transsignalling pathways. While classical signalling is associated with the anti-inflammatory activities of the cytokine, the IL6 trans-signalling pathway mediates chronic inflammation and is therefore a target for therapeutic intervention. Extracellular vesicles (EVs) are natural, lipid-bound nanoparticles, with potential as targeted delivery vehicles for therapeutic macromolecules. Here, we engineered EVs to express IL6 signal transducer (IL6ST) decoy receptors to selectively inhibit the IL6 trans-signalling pathway. The potency of the IL6ST decoy receptor EVs was optimized by inclusion of a GCN4 dimerization domain and a peptide sequence derived from syntenin-1 which targets the decoy receptor to EVs. The resulting engineered EVs were able to efficiently inhibit activation of the IL6 transsignalling pathway in reporter cells, while having no effect on the IL6 classical signalling. IL6ST decoy receptor EVs, were also capable of blocking the IL6 trans-signalling pathway in C2C12 myoblasts and myotubes, thereby inhibiting the phosphorylation of STAT3 and partially reversing the anti-differentiation effects observed when treating cells with IL6/IL6R complexes. Treatment of a Duchenne muscular dystrophy mouse model with IL6ST decoy receptor EVs resulted in a reduction in STAT3 phosphorylation in the quadriceps and gastrocnemius muscles of these mice, thereby demonstrating in vivo activity of the decoy receptor EVs as a potential therapy. Taken together, this study reveals the IL6 trans-signalling pathway as a promising therapeutic target in DMD, and demonstrates the therapeutic potential of IL6ST decoy receptor EVs.

molecular biology↗

bioRxiv2020

Impacts of radiation on the bacterial and fungal microbiome of small mammals in the Chernobyl Exclusion Zone

Environmental impacts of the 1986 Chernobyl Nuclear Power Plant accident are much debated, but the effects of radiation on host microbiomes has received little attention to date. We present the first analysis of small mammal gut microbiome from the Chernobyl Exclusion Zone in relation to total absorbed dose rate and including caecum as well as faeces sample. The associations between microbiome communities and radiation exposure varied between host species. Associations between microbiome and radiation was different for analyses based on ambient versus total weighted absorbed dose rates. We found considerable variation between patterns for faecal and gut samples of bank voles, suggesting faecal samples are not an accurate indicator of gut composition. For bank vole guts, associations between radiation and bacterial community composition were robust against geographical and habitat variation. We found limited associations between radiation and fungal communities. Host physiological mechanisms or environmental factors may be driving these patterns.

ecology↗

bioRxiv2020

Cholesterol metabolism drives regulatory B cell function

Regulatory B cells restrict immune and inflammatory responses across a number of contexts. This capacity is mediated primarily through the production of IL-10. Here we demonstrate that the induction of a regulatory program in human B cells is dependent on a metabolic priming event driven by cholesterol metabolism. Synthesis of the metabolic intermediate geranylgeranyl pyrophosphate (GGPP) was required to specifically drive IL-10 production, and to attenuate Th1 responses. Furthermore, GGPP-dependent protein modifications controlled signaling through PI3K{delta}-AKT-GSK3, which in turn promoted BLIMP1-dependent IL-10 production. Inherited gene mutations in cholesterol metabolism result in a severe autoinflammatory syndrome, termed mevalonate kinase deficiency (MKD). Consistent with our findings, B cells from MKD patients induced poor IL-10 responses and were functionally impaired. Moreover, metabolic supplementation with GGPP was able to reverse this defect. Collectively, our data define cholesterol metabolism as an integral metabolic pathway for the optimal functioning of human IL-10 producing regulatory B cells. Graphical abstractMKD = Mevalonate kinase deficiency O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/893982v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@e23fd0org.highwire.dtl.DTLVardef@1cb9611org.highwire.dtl.DTLVardef@e9769corg.highwire.dtl.DTLVardef@16a1d0f_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗