NIHRDataInsights/HRCSData huggingface dataset:
Title: Nudc as a new molecular target to investigate the pathogenesis and treatment of skeletal ciliopathies.
Funder: Medical Research Council

Abstract: Key aims of this proposal are: 1) To characterise the protein-protein interaction between IFT80 and NUDC: Having identified a novel interaction between IFT80 and NUDC using tandem affinity purification (TAP), we will now confirm this interaction using co-immunoprecipitation (co-IP) experiments in various ciliated and non-ciliated cells lines, using full length and also deletion constructs for both proteins, to characterise the particular domains mediating this interaction. 2) To systematically identify NUDC-interacting proteins: We will now use further TAP experiments, this time using NUDC as bait, to identify the full repertoire of NUDC-interacting proteins. We will then confirm any novel interactions identified in this screen using further co-IP experiments. 3) To analyse the function of IFT80- and NUDC-interacting proteins in zebrafish: In order to evaluate the significance of these protein-protein interactions in a vertebrate model of ciliopathies, we will use morpholinos to knock-down genes encoding novel interactors in zebrafish and assess ciliopathies and other phenotypes, as well as cilia formation and function. Genetic interactions between these and other intraflagellar transport proteins will also be evaluated using pairwise morpholino injections. 4) To generate and characterise a Nudc mouse mutant: Using ES cells that have been successfully targeted for Nudc, we will generate mice carrying a mutation in this gene. These mice will be fully characterised for ciliopathy phenotypes and cilia formation. We will also generate primary mouse embryonic fibroblasts for cell biological and immuonflourescence studies. 5) To test for rescue of ciliopathies following knockdown of Nudc: We will cross Nudc or Ift80 mutant mice with Dync2h1 mutant mice to test for genetic rescue of ciliopathies. This will provide proof-of-concept in terms of targeting dynein trafficking for therapy. MEFs from these mice will also be analysed for ciliary trafficking defects.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: MUSCULOSKELETAL
Title: Investigation of the Structural Quality of Bone and Cartilage Repair Following Cell Therapy
Funder: Medical Research Council

Abstract: We aim to study the structural quality of bone and cartilage repair tissue in degenerate or injured knee joints following cell therapy with either chondrocytes and/or mesenchymal stromal cells. We have already been granted funding by Arthritis Research UK to carry out a randomised controlled trial in 114 subjects to compare the clinical benefit of the three cell therapy strategies (Autologous Stem cells, Chondrocytes Or the Two, ASCOT). The work described in this proposal would require the subjects recruited into the ASCOT trial to undergo 3 additional sets of investigations: imaging, arthroscopy and histological assessments at approximately 1 year after the cell implantation. The main objectives of this 'add-on' study would be (1) To determine the structural quality of repair tissue which forms in these patients, using MRI/CT, visual assessment and histology (including immunohistochemistry). This will allow us to look at a whole range of outcomes including: repair cartilage thickness, volume, signal intensity, surface integrity, morphology and biochemical content and subchondral bone structure including the presence of inflammation, oedema and cysts, determination of cortical integrity, bone densitometry, macroscopic appearance of the repair site and the adjacent tissues. (2) To relate structural quality of repair to the clinical outcome of the patients, the characteristics of the cells being implanted and the environment they will be implanted into (cell characterisation and biomarker analysis funded elsewhere). (3) To investigate whether MRI or CT can be used in aiding the selection of patients who will benefit from ACI. (4) To trial an FT-IRIS probe to investigate the integrity, composition and organisation of the repair tissue in a less invasive way than by biopsy. (5) To help to elucidate which cell types are most appropriate for treating joints with osteochondral defects or osteoarthritis.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: MUSCULOSKELETAL
Title: FEMORAL: Factors affEcting MortALity, morbidity and patient outcomes after joint replacement surgery.
Funder: Medical Research Council

Abstract: Background: Osteoarthritis in the elderly is very common in the UK and around the world, and can result in a significant reduction in mobility, quality of life and increased economic burden. Joint replacement surgery (JRS) is one method of treating osteoarthritis, but analysing and interpreting data arising from the NHS presents specific challenges. The organisational structure of the NHS gives rise to a dependent structure of data. The hierarchical structure of organisational data is commonly represented using a traditional multi-level model, but in practice violations of this strict hierarchy occur, as surgeons and patients move between surgical units and surgeons respectively. Aims: We aim to improve patient outcomes and reduce mortality and morbidity after JRS by identifying factors which affect the efficacy of JRS. Methods: Using data from the National Joint Registry we aim to explore the aims and objectives of the fellowship by employing multiple membership multi-level modelling techniques. Features of the prosthetic implant and surgeon preference will be incorporated into the modelling strategy to help understand the choices surgeons make for their patients. Longitudinal changes will be investigated allowing changes in surgeon or surgical unit performance be identified. Models will take into account patient characteristics, and the performance of prosthesis, surgeons and surgical units can all be considered conditionally on the patient. Scientific/Medical opportunities: Our findings will provide novel insights into factors affecting the efficacy of JRS; it will be enable researchers to identify features of prosthesis and surgical approaches which optimize patient outcomes. It will facilitate the Identification of high and low performing surgeons and surgical units to ensure the dissemination of good practice and resolution of poor practice. Our findings will allow surgeons to tailor their selection of prosthetic joints to individual patients.


Research activity codes: 6.4 SURGERY;7.3 MANAGEMENT AND DECISION MAKING
Health categories: MUSCULOSKELETAL
Title: Investigating phosphatidylserine metabolism in Lenz-Majewski syndrome
Funder: Medical Research Council

Abstract: We aim to understand the biochemical interaction between disordered phosphatidylserine (PS) synthesis and bone metabolism as highlighted in Lenz-Majewski syndrome (LMS). We will employ UPLC tandem mass spectrometry to investigate lipid composition and the production of specific PS isoforms, first in patient fibroblasts and lymphoblasts cell lines and later in cells derived from animal models. Biochemical analysis will be performed on osteoclasts derived from LMS whole blood. We will investigate PS metabolism in mice with reduced PS synthase enzyme activity and create knock in mice mimicking the common human mutation. This will use standard methods including homologous recombination in ES cells and chimera production. Zebrafish mutants will be created using the Tol2 kit. This method facilitates the rapid construction of mutants allowing insertion or replacement of different elements in a modular form e.g. promoters or protein tags. We will use different promoters to drive expression in specific cell lineages to dissect which are relevant for PS in disordered bone development. Osteogenesis and later skeletal development will be investigated in mice and fish using standard in situ hybridization and histological techniques. Postnatal skeletal analysis on mice up to 1 year old will be monitored using DEXA and microCT, the latter also used to investigate the fish skeleton. We will use the most appropriate model system to test the ability to modify PS biosynthesis using molecules or compounds that might have therapeutic benefit. It is likely that this will be a patient, fish or mouse cell line grown in 384-well microtitre plates in the presence of deuterated stable isotopes derivatives of fatty acids/serine. Cells can then be assayed following treatment with an array of test molecules, using UPLC tandem mass spectrometry. This final aim is specifically relevant clinically for treatment of LMS patients and potentially in more generalised bone disease.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: MUSCULOSKELETAL
Title: Periodic paralysis: from molecules to mice
Funder: Medical Research Council

Abstract: Periodic paralyses (PPs) are a group of rare muscular disorders with episodes of flaccid muscle weakness. Episodes are associated with low (Hypokalaemic PP (HypoPP)) or high (Hyperkalaemic PP (HyperPP)) serum potassium. HyperPP can present with or without interictal myotonic stiffness. The syndromes overlap with Paramyotonia Congenita (PMC). In addition, many PP patients develop a disabling myopathy later in life. Genetic studies of PP patients have identified mutations affecting genes that code for the skeletal muscle ion channels Nav1.4 and Cav1.1. This proposal is based on our ongoing efforts to study the functional effects of these mutations, and to implement three experimental strategies to significantly improve the characterization of the pathophysiology of the PPs and of the effects of targeted therapies. 1. In order to improve the molecular characterization of the effects of PP mutations we will employ Cut-open voltage clamp (COVC) for measurements of gating pore and gating currents in Xenopus oocytes. 2. In order to express Cav1.1 channels that are difficult to express heterologously, we will use a muscular dysgenesis mouse cell line GLT. This myotube-based expression system will allow us to study the effects of mutant channels on myotube membrane excitability. Molecular and cellular properties of patient derived myotubes will also be investigated. 3. In order to study the mechanisms of permanent muscle weakness we will employ a novel mouse model of PP with myotonia and will characterize changes in electrophysiological properties of muscle fibres in permanently weak muscles. For each aim we will include an investigation of the effects of therapeutic agents to intervene in the detected pathomechanisms in order to develop mutation specific, personalized therapies.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;5.2 CELLULAR AND GENE THERAPIES
Health categories: MUSCULOSKELETAL
Title: Role of motor unit remodelling in age-related loss of skeletal muscle fibres.
Funder: Medical Research Council

Abstract: The causes of age-related loss of muscle fibres are unknown, but it is associated with remodelling and loss of motor units. Studies will use novel state-of-the-art approaches to determine how motor unit remodelling (stimulated by crush of the common peroneal nerve and subsequent re-innervation in mice) influences activation of 3 key degenerative pathways (increased hydrogen peroxide, increased pro-inflammatory cytokines, activation of FOXO-dependent atrophic processes) in single muscle fibres in vivo and whether these processes contribute to age-related loss of muscle fibres and weakness. Intra vital imaging will be used to study the structure of peripheral axons and NMJ in superficial single muscle fibres from thy1-CFP (cyan fluorescent protein) mice expressing CFP in all neurons. Activation of the 3 key degenerative pathways will be examined in the same single muscle fibres transfected with GFP/YFP-based probes using AAV vectors. Studies will examine whether transient denervation of muscle fibres in adult mice leads to activation of these 3 key degenerative pathways in fibres and whether this is confined to fibres with compromised innervation or also occurs in innervated fibres. Studies in old mice will determine whether activation of the 3 key pathways occurs in all fibres or only in those fibres with compromised innervation and will also examine the effect of nerve crush and regeneration on the axons and NMJ of muscles of old mice. In parallel studies, neuromuscular transmission in single fibres will be examined to determine whether changes in NMJ structure in old mice prevent neuromuscular transmission and the relationship of this to activation of degenerative pathways in the fibre. Finally, we will determine whether degenerative pathways activated in denervated muscle generate muscle-derived axonal sprouting factors, and muscle mRNA will be analysed to identify potential muscle-derived axonal sprouting factors that are attenuated by ageing.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: MUSCULOSKELETAL
Title: Progressive Muscular Weakness in Periodic Paralysis and Normal Ageing
Funder: Medical Research Council

Abstract: 1. Normal age-related changes in the skeletal muscle 'channelome', muscle structure and excitation-contraction (EC) coupling EDL and soleus of wild-type male mice (3,12,18,24 months) and quadriceps muscle from normal males (18-33, 34-49, 50-64, 65+) will be used to examine age related changes in: a) Muscle strength and EC coupling using muscle tension testing on explanted mouse EDL and soleus. Supramaximal electrical stimulation will be compared with supra maximal activation with caffeine. b) The skeletal muscle channelome using exon level RNA sequencing (mouse & human muscle) c) Muscle structure and ultrastructure will be examined using light and electron microscopy (mouse & human muscle) d) Expression and post translational modification of proteins involved in excitation and EC coupling (i.e. voltage-gated sodium channel, calcium channel and ryanodine receptor) using immunohistochemistry (mouse & human muscle) 2. PP age-related changes in skeletal muscle 'channelome', muscle structure and EC coupling HyperPP mouse muscle and muscle from male patients with HypoPP (Cav1.1 R528H or R1239H) and HyperPP (Nav1.4 T704M or M1592V) will be used. Muscle will be processed and examined as for Aim 1. 3. The effect of prolonged depolarisation on muscle and how it varies with age To model an acute attack of PP we will induce prolonged depolarisation by exposing normal mouse muscle of different ages to either barium or gramicidin and low potassium solution, or Hyper PP mouse muscle of different ages to high potassium solution. We will then examine: a. if EC uncoupling or established histological features of PP (i.e. vacuoles) occur after prolonged depolarisation and how age effects this b. if age related changes occur in younger muscle after an episode of prolonged depolarisation c. If there is a final common pathogenic pathway following prolonged depolarisation in vitro d. If pathogenic changes can be minimised with certain pharmacological agents.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: MUSCULOSKELETAL
Title: Understanding and Preventing Inappropriate Osteogenic Differentiation
Funder: Medical Research Council

Abstract: Heterotopic ossification (HO) is frequently observed in individuals after traumatic injury. The cell types that inappropriately differentiate into osteoblasts, in addition to the molecular mechanisms underlying HO remain elusive. Current research approaches often utilise bone marrow stem cells, and evaluate how they differentiate into osteoblasts. In this project, we rationalised that since HO is regarded as inappropriate differentiation, we should use a cell type that can, but shouldn't turn into bone in normal circumstances. We settled on using hair follicle dermal papilla cells, which in the correct culture conditions can inappropriately differentiate into bone. In Objective 1 we will utilse a modified shock tube to mechanically injure dermal papilla cell cultures. We aim to establish the conditions whereby mechanical injury can drive osteogenesis, assessing whether the cells themselves provide all the signals necessary to drive differentiation. In Objective 2, we will use ChIP-Seq to identify the location of Ets1 and Runx2 transcription factor (TF) binding sites. Ets1 and Runx2 are two TFs known to bind DNA and drive osteogenesis. We will determine whether these binding sites are in active, poised, or repressed states, comparing fibroblasts, and papilla cells, both before and after they have been injured using the shock tube. This will resolve if papilla cells have TF binding sites that are always open, or whether injury elevates these sites into an active state, resulting in gene expression. In Objective 3 we establish a reporter screening assay that will express GFP when papilla cells are pushed to an osteoblast fate. We will utilise this assay to detect small molecules and proteins that can inhibit inappropriate osteoblast differentiation in dermal papilla cells. Completion of this work will provide new insights into inappropriate osteoblast differentiation, which is a fundamental requirement if we aim to prevent HO.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: MUSCULOSKELETAL
Title: Myosin-linked mechanisms for the regulation of muscle contraction
Funder: Medical Research Council

Abstract: This project aims to elucidate the molecular mechanisms of thick filament-based regulation in skeletal muscle. These mechanisms supplement the well-known thin filament-mediated pathways to control the strength and duration of contraction in response to muscle length, load and previous activity, making them attractive targets for therapeutic intervention in diseases of both skeletal and cardiac muscle. Using bifunctional fluorescent probes, we will determine the in situ conformation of the regulatory light chain (RLC) region of myosin in the OFF state of the thick filament. We will test the hypothesis that the RLC N-terminal lobe is primarily a regulatory filament-docking domain rather than, as in current models, an extension of the lever arm of the myosin motor. We will characterize the changes in RLC conformation associated with the OFF-ON transition of the thick filament, and investigate how that transition is controlled by the nucleotide state of myosin and the inter-filament spacing. We will determine how RLC conformation is controlled by calcium, both in the steady state at intermediate calcium concentrations and kinetically following a calcium jump, and thus elucidate the relationship between calcium activation of the thin filament and the OFF-ON transition in the thick filament. We will characterise the changes in RLC conformation produced by RLC phosphorylation, and relate them to the increased force output associated with previous muscle activity. We will determine the dependence of RLC conformation on the static and dynamic stress in the thick filament in order to elucidate the role of mechano-sensing in muscle regulation. Using N-terminal fragments of myosin binding protein C, we will determine the role of this protein in signalling between thin and thick filaments. Combining these results, we will produce a molecular model of the physiological regulation of contractility of skeletal muscle that integrates thin and thick filament-based mechanisms.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: MUSCULOSKELETAL
Title: Determining how mutations in actin lead to skeletal muscle weakness
Funder: Medical Research Council

Abstract: Importance and main objective: Diseases related to mutations in actin are characterized by severe skeletal muscle weakness often leading to premature death. The understanding of this group of disorders has advanced in recent years through the identification of the causative ACTA1 gene mutations. Nevertheless, the exact mechanisms leading to muscle dysfunction remain unclear, which hampers the development of efficient therapies. Therefore, the main objective of the present research project is to clearly characterise the cascade of molecular and cellular events triggering muscle wasting. Methods: We will mainly use muscle samples and isolated myofibres from two transgenic mouse models perfectly recapitulating the human condition and expressing either His40Tyr or Asp286Gly single amino acid substitution in actin. We will then perform a broad range of experiments including small-angle X-ray scattering, polarised fluorescence, myofibre mechanics, confocal microscopy, immunohistochemistry and micro-array analysis. Feasibility and outcomes: The availability and knowledge of these unique techniques in combination with experience in undertaking challenging studies put us in a unique position to tackle the problem posed. We expect to demonstrate that mutations in actin directly impair actin filament extensibility, inducing a disruption (i) of myosin binding and (ii) of the intrinsic force-generating capacity, directly promoting myofibre atrophy. Successfully proving this would grandly facilitate the design of efficient therapeutic interventions that would target the actin-myosin interface.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: MUSCULOSKELETAL
Title: MICA: The role of utrophin in DMD and its therapeutic potential
Funder: Medical Research Council

Abstract: Duchenne Muscular Dystrophy (DMD) is an X-linked recessive muscle wasting disorder. Patients are wheelchair-bound by the age of 12 and die from respiratory failure or cardiomyopathy in their twenties. DMD is caused by the absence of the cytoskeletal protein dystrophin at the sarcolemma which makes it more susceptible to contraction-induced injury. There is currently no effective treatment for DMD. The strategy we are taking is to modulate the expression of the dystrophin related protein utrophin which can compensate for the missing dystrophin. This approach is applicable to all patients whatever their mutation. We will identify lead candidate small molecules for the modulation of utrophin expression by high through-put screening of dystrophin deficient immortalised mouse muscle cell lines. Our initial work has led to a first-in-class agent, SMT C1100, for the pharmacological modulation of utrophin which is entering a Phase Ib clinical trial. However, we now need to identify and develop best in class compounds to improve on current drug effectiveness. We will optimise any hits from the cell lines using medicinal chemistry and in human DMD cell lines. We will then assess lead candidates in vivo in the mdx mouse model of the disease. Promising candidate molecules will be assessed for their effect on the muscle pathology using histopatholgical and molecular analysis, assays of eccentric contraction in EDL muscle and MRI studies of the heart. We will analyse biomarkers for the disease such as miRNAs, FN1 and MMP9. We will also determine the mechanism of action of drug candidates which may allow the development of other molecular targets. Our aim is to have generated lead drug candidates that by the end of the five years of this basic preclinical work can be taken into the clinic by Summit Therapeutics.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: MUSCULOSKELETAL