NIHRDataInsights/HRCSData huggingface dataset:
Title: Formation and function of Membrane Contact Sites between the ER and the phagocytic pathway in the Retinal Pigment Epithelium.
Funder: Medical Research Council

Abstract: The retinal pigment epithelium (RPE) phagocytoses daily shed photoreceptor outer segments (POSs). Little is known of how POS-derived membrane lipid is processed; considerable processing is required for secretion to the choroid, including cholesterol esterification in the ER, suggesting the existence of phagosome:ER lipid transport mechanisms. The ER forms membrane contact sites (MCSs), microdomains of close membrane apposition (<30nm), with a diverse range of organelles. We have previously used electron microscopy (EM) to identify membrane contact sites (MCSs) between the ER and endocytic pathway in Hela cells. We have identified regulators of MCS formation and established their role in endocytic pathway:ER transport of LDL-derived cholesterol. We and others have observed ER:phagosome MCSs and I now propose to investigate their regulation and function in the RPE, taking advantage of unique EM tools I have developed. I will first characterise the extent of ER:phagosome MCSs in macrophages and cultured RPE cells fed isolated POSs as well as in mouse eye tissue. I will then examine MCSs in macrophages and RPE cells depleted of regulators of ER MCSs with the endocytic pathway, or of STIM1, a phagosome-specific candidate regulator. I will additionally screen for novel MCS regulators, as well as determining the effect of manipulating cytosolic calcium concentrations on ER:phagosome MCS formation and phagosome maturation. The physiological relevance will be tested in eyes isolated from mouse models where available. Having established key ER:phagocytic pathway MCS regulators, I will measure the effects of MCS manipulation on lipid transport and cholesterol esterification in cultured RPE fed with isolated POSs. This project will elucidate the potential role of MCSs in the fundamental process of the transport and esterification of POS-derived free cholesterol in the RPE for secretion of lipoproteins, with potential significance to the development of AMD.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: EAR
Title: Biofabrication and characterisation of a new class of functional and durable auricular cartilage implants
Funder: Medical Research Council

Abstract: Introduction: Patients undergoing auricular reconstruction for defects following cancer and trauma require long- term, stable repair. Current methods involve autologous cartilage harvest, which is has limited availability and is associated with donor site morbidity. The current generation of tissue engineered implants do not produce stable cartilage resistant to uncontrolled shrinkage, resulting in suboptimal reconstruction. Our objective is to combine the use of tissue specific stem cells, novel biofabrication techniques and targeted maturation to engineer a new class of functional yet durable cartilage implants that can overcome these limitations. Methods: Phase I - Human auricular cartilage stem cell isolation (ethics approved), storage, proliferation analysis, characterisation (FACS, Ab, qPCR) Phase II - Chondrogenic differentiation of stem cells (chondrogenesis, osteogenesis, adipogenesis, vasculogenesis), optimise chondrogenic medium (iCelligence), pellet cultures, optimal bio-ink for auricular stem cells (hydrogel vs. type II collagens), compare with another MSC (i.e. ADSC) Phase III - 3D Bioprinting (EnviscoTec Bioplotter) of auricular chondroprogenitor cells with bioinks Phase IV - Growth factor-induced in vitro maturation of auricular constructs, biomechanical and biochemical testing of mature constructs, comparison with native cartilage Discussion: Through pilot studies, we demonstrated cellular, biomechanical and morphological differences between mature and immature bovine auricular cartilage. Our group has previously demonstrated precocious maturation of immature articular cartilage using FGF2 and TGb1. We isolated and cultured auricular tissue-specific stem cells proving feasibility. Exciting advances such as maturation and 3D bioprinting make us uniquely placed to recreate native microarchitecture and form functional cartilage with real translational potential that has wide-ranging implications for tissue-engineering, beyond the auricle.


Research activity codes: 5.4 SURGERY
Health categories: EAR
Title: Interaction between sensory and supporting cells in the organ of Corti: basis for sensitivity and frequency selectivity of mammalian cochlea.
Funder: Medical Research Council

Abstract: Our prime objective is to study interaction between outer hair cells (OHCs) and the supporting cells (SCs) that comprise a restraining, complex, flexible, fluid filled cage that is proposed to optimise exchange and control of energy between OHCs and the cochlear partition, including the basilar membrane (BM). Cochlear HCs die and are not replaced when damaged by exposure to intense sounds, disease, age, and genetic disorders. Recently, however, we have shown that SCs can be converted into HCs at various postnatal stages, but remain immature. HC immaturity is likely due to lack of interaction with surrounding SCs, which is why it is essential to understand this interaction for restoration of hearing. To this end, we will systematically modify and delete the motor protein, prestin, in OHCs and cytoskeletal proteins in SCs in mice and produce mouse models of age-related and congenital hearing loss. With mice that express channel rhodopsins in OHCs and SCs, we can excite and reversibly change the mechanical properties of the cochlea with light flashes. Through modelling, based on in vivo and in vitro acoustical, mechanical, and electrical measurements, our understanding of the functional significance of interaction between OHCs and their SC cages can be developed and tested, leading to the detailed understanding necessary to fully exploit the exciting regenerative possibilities now becoming available. SCs, but not HCs, are interconnected by gap junctions that are attributed with roles in fluid homeostasis and/or intercellular signalling. Connexins 26 (Cx26) and CX30 gap junctions, which interconnect SCs, have also been implicated in the transmission of forces within the OC. Most hereditary hearing disorders, including age-related hearing-loss (ARHL) are associated with defects in CX26 and CX30 expression. We aim to discover how Cx26 and Cx30 mutations alter cochlear mechanical properties and the Cx30 mutation rescues hearing loss in a mouse strain with severe ARHL.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;5.1 PHARMACEUTICALS
Health categories: EAR
Title: Cochlear stress granules - a therapeutic target for auditory protection?
Funder: Medical Research Council

Abstract: The WHO estimates that 360 million people worldwide have a disabling hearing loss. A primary cause of this sensory deficit is permanent loss of sensory receptor hair cells resulting from exposure to noise or ototoxic drugs, infection, or through ageing. No medicines are available to treat hearing loss, and hearing aids fails to provide a benefit for the majority (~90%). This leaves a clear, unmet need for a biological-based therapy that prevents hearing loss by preventing hair cell death. Stress granules are cytoplasmic complexes of mRNA and RNA-binding proteins that form in cells undergoing stress. We have shown that stress granules form in cochlear hair cells after exposure to ototoxic drugs or noise. Our hypothesis is that stress granules play an important role in sensory hair cell survival. This project takes three approaches to test this: (1) Conditional gene deletion will be used to determine the contribution of two stress granule components, TIA-1 and Caprin-1, to hair cell survival in normal hearing and when the cochlea is challenged by acoustic or ototoxic trauma. Auditory system function will be assessed by auditory brainstem response measurement, combined with histological and immunohistochemical assessment of cell survival, stress granule formation and protein constitution. (2) Cochlear explant models will be used to test the effect of drugs that can enhance or inhibit stress granule formation on hair cell survival. Drugs that increase hair cell survival will then be further studied in animal models. (3) Conditional RiboTag mice and RNAseq will be used to determine the effects of trauma on the translational profile of sensory hair cells in vivo, and after conditional deletion of stress granule components. The research will determine whether stress granules can regulate hair cell survival, and it has the potential to generate novel therapeutics for the protection of those sensory cells and thus the prevention of hearing loss.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;5.1 PHARMACEUTICALS
Health categories: EAR
Title: Sphingosine-1-phosphate signalling in hearing loss
Funder: Medical Research Council

Abstract: We know that sphingosine-1-phosphate (S1P) signalling is required for hearing because mutations in three genes in this pathway lead to progressive deafness in the mouse. Analysis of these mice suggests that maintenance of the normal electrochemical environment of the fluid bathing sensory hair cells of the cochlea is at fault. The main objective is to ask if manipulating S1P signalling can reverse hearing loss as a proof-of-principle. The specific questions are: 1. What are the mechanisms underlying progressive hearing loss in mice with mutations affecting S1P signalling? 2. Can progressive hearing loss in S1P-associated deafness be halted or reversed? 3. Is S1P signalling involved in deafness resulting from other triggers for hearing loss? The first question will be addressed by detailed analysis of the time course of structural and physiological changes in two of the affected lines of mouse (one with rapid and one with slower progression of hearing loss) and identification of the cell types within the cochlea that require S1P signalling using a genetic approach to target gene deletion to specific cells and at specific ages. These findings will tell us which cells we need to target for treatment and when. The second question involves using a genetic approach to restore normal gene function before, during and after the onset of hearing loss to ask if it can be prevented, halted or even reversed after it has become established. These experiments will aim to determine a critical period for treatment to avoid hearing loss. The third question will bring in a mouse line with a defect in maintenance of the cochlear fluids due to a different cause, unrelated to S1P. In this mouse line, S1P signalling will be manipulated using genetics to ask if boosting S1P activity can improve hearing irrespective of the specific trigger. If so, then manipulating S1P signalling pharmacologically may be beneficial in a much broader range of cases of human deafness.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;5.1 PHARMACEUTICALS
Health categories: EAR
Title: Investigating the etiology of cholesteatoma
Funder: Medical Research Council

Abstract: Cholesteatomas are invasive middle ear cysts. Acquired cholesteatomas appear to form from retraction pockets in the tympanic membrane and several theories have been suggested to account for their initiation. These cysts form associated with very specific regions of the tympanic membrane, suggesting some underlying developmental differences. We aim to investigate the development of the tympanic membrane, the mechanics of different parts of the membrane and the difference between cholesteatomas that form in different areas of the membrane, using a range of cutting edge techniques in mouse and human. Aim1: To understand the development of the pars flaccida and pars tensa during mouse and human development Aim 2: To understand differences in mechanical properties of the murine tympanic membrane in different parts of the ear in culture Aim 3: To identify differences between cholesteatomas that form in different parts of the ear Methods: Transgenic reporter mouse lines will be used to trace the contribution of cells of different tissue origin, and of specific structural genes. Ears will be imaged using confocal and multiphoton microscopes and using optical projection tomography to generate 3D images. Tissue culture of tympanic rings will be used to assess different properties of the tympanic membrane, using time-lapse photography to follow cell movement. Lastly RNAseq will be utilised to identify differences between cholesteatomas that develop in different parts of the ear. This research is driven by recent advances in the field of ear development, which open up a whole new previously unappreciated avenue of research. The proposed research is novel and directly related to understanding the underlying causes of disease. All the mouse lines needed are available and the questions asked are driven by real clinical problems. The proposal is likely to make clear advances in the field, helping to understand the mechanisms behind a debilitating ear problem.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: EAR
Title: IMPC: The role of SARNP in hearing loss
Funder: Medical Research Council

Abstract: DNA resides within the nucleus in human cells and encodes the information to make proteins. Messenger RNA (mRNA) is copied from DNA and is then transported to the cytoplasm where it is translated to make proteins. A key protein complex involved in the transport of mRNA from the nucleus to the cytoplasm is TREX. We previously identified SARNP protein as a subunit of the TREX mRNA export complex which forms an ATP dependent trimer with two other subunits, ALYREF and UAP56 and plays a role in mRNA export. Mutations in TREX subunits are associated with a number of human conditions including X-linked intellectual disability (THOC2) and Beaulieu-Boycott-Innes syndrome (THOC6). Although the preliminary phenotypic screening of a SARNP knockout heterozygote has identified a hearing defect, the molecular and functional mechanisms controlled by this protein in the auditory system are completely unknown. In this project we will combine our expertise in mRNA export mechanisms with that in hearing research to identify the cellular defects leading to hearing loss in these mice.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: EAR
Title: Epigenetic mechanisms underlying hearing impairment
Funder: Medical Research Council

Abstract: Hearing loss affects many people and is often due to hair cell (HC) loss in the cochlea. HCs cannot regenerate, while surrounding supporting cells (SCs) can undergo reprogramming to form new HCs, but this ability is lost shortly after birth. Major questions are: how to promote HC regeneration from endogenous sources and how to prevent HC and SC degeneration? Increasingly associated with disease and reprogramming, epigenetic changes are likely to play a crucial role. We will study the chromatin remodellers Chd7 and -4, which when mutated in humans cause sensorineural hearing loss. CHD7 mutations are associated with CHARGE and other syndromes, while mutations in CHD4 cause CHARGE-like defects. Our data in mice indicate that without Chd7 hair cells die. This project will unravel the molecular function of Chd7 and -4 in the mouse ear to identify cell-specific targets and elucidate their mechanism by i) deleting Chd7/4 in hair and supporting cells to characterise their phenotype ii) determining the time when Chd7 function in vivo can be rescued iii) examining the transcriptome changes in Chd7/4 mutant and control HCs and SCs at specific time points Integration of these data is a powerful strategy to identify direct targets unique and in common to Chd4 and Chd7 and thus link hearing loss due to Chd7/4 mutations with downstream molecular events. These data provide a rich resource to use bioinformatics and modelling to unravel the gene networks downstream of Chd7 and -4 as the basis to discover new causative deafness genes and pathways. This research will not only elucidate the mechanism of sensorineural hearing loss in CHARGE and other syndromes, but also form the basis for restorative strategies. It will provide a road into understanding the epigenetic landscape of the normal and degenerating ear and thus have wide implications to develop regenerative or protective strategies to restore hearing.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: EAR
Title: Analysis of the tissue and temporal specific role of Eya1 in ear development and its contribution to hearing loss
Funder: Medical Research Council

Abstract: This project involves use of a conditional Eya1 mouse to follow the role of this gene in specific compartments of the ear during development. Eya1 has a complex expression pattern spanning the whole of embryonic ear development, from E9.5 to postnatal stages with expression in the endoderm, ectoderm, neural crest and mesoderm of the craniofacial region. Using different Cre drivers we can address key questions regarding the role of Eya1 at different time points and in different tissues. In this proposal we will concentrate on the role of Eya1 in the neural crest (Wnt1cre), the endoderm (Sox17 -2a-icre), and the mesoderm (Mesp1cre), and use the PCagErt2cre line to investigate the relative role of Eya1 during key stages of ear development, from the formation of the otic vesicle and the pharyngeal arches to the cavitation of the middle ear at birth. Together this will allow us to dissect out the role of Eya1 and provide an understanding of the defect observed in patients with Eya1 mutations.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: EAR
Title: Towards the development of a prognostic tool for cochlear implant outcome using functional near-infrared spectroscopy (fNIRS).
Funder: Medical Research Council

Abstract: Background: Cochlear implants (CIs) allow many children to develop speech at an age-appropriate level when deafness is recognised early in life. However, the ability to perceive speech in children with CIs still varies considerably. Early identification of children at risk of developing poor speech perception is crucial to maximise the benefit of any rehabilitation approach. However, current measures of speech perception ability require behavioural assessments. Since very young children are difficult to assess with behavioural techniques, there is often a delay of many years between implantation and the detection of poor speech perception. In order to predict speech performance in children with a CI, it is necessary to find a suitable objective measurement of speech perception at an early age. Aims & objectives: The aim of the proposed work is to evaluate whether we can predict good and poorer CI performance using brain imaging before and shortly after cochlear implantation. We will measure brain activity in infants using fNIRS, an optical brain-imaging technique that is fully compatible with a CI. We hypothesise that the benefit of a CI will be associated with cortical plasticity to auditory and visual speech, which may form the basis for a prognostic marker of CI outcome. Methodology: Paediatirc CI candidates (age at implantation 6 months to 2 years old) will be undergo fNIRS measurements i) 1 month before CI surgery ('pre-implantation'), ii) 1 month after activation of CI ('early post-implantation') iii) 12 months after activation of CI in addition to behavioural speech perception measurements ('follow up'). Scientific and medical opportunities of the study: A prognostic tool could better inform parents on the likely treatment outcome for their child and also enable clinicians to tailor rehabilitation and signal processing strategies to an individual patient's needs. This study will inform us of mechanisms of cortical plasticity in children after implantation.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: EAR
Title: Understanding and enhancing repair of the tympanic membrane
Funder: Medical Research Council

Abstract: Ear drum perforations have been studied in a number of animal models. In each of the cases with animal models the perforation has been performed in vivo, with the healing followed up in a number of different animals. No live imaging of healing has been described due to the problems of accessing the ear. In this proposal we introduce a novel explant culture method in the mouse allowing accurate perforations to be made outside of the mouse and the healing process to be followed in situ in the same ear. This technique also allows us to take advantage of a suite of transgenic mice to follow clones of cells and specific stem/progenitor populations to address previously unanswerable questions. It also avoids the use of multiple animals sacrificed at different time points and invasive in vivo surgery. To track cells live in culture we will use TdTomato transgenic mice crossed to a range of Cre reporter mice: putative stem cells and progeny (Sox2creERT2), neural crest (Wnt1cre), Epithelium (K14cre and K14creERT2), Endoderm (Sox17icre), mesoderm derived vasculature (Mesp1cre), combined with the use of DiI and DiO. To follow changes in proliferation in specific tissues during repair we will utilise R26Fucci2A mice, crossed to these various Cres. To perturb cellular processes (proliferation, vascularisation, cytoskeleton arrangement) we will add various small molecule inhibitors to the culture system. To understand how the putative stem cells niche is controlled we will investigate the role of Wnt signalling, through the use of pharmacological stimulators and inhibitors of this pathway and through the use of transgenice mice where the pathway is stimulated (Axin2lacZhom, B-catenin gain of function) or repressed (Wntless/GRP177). Finally we will utilise our model to focus on understanding the mechanisms behind the current clinical use of Fgf ligands to treat perforations.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: EAR