NIHRDataInsights/HRCSData huggingface dataset:
Title: Investigating the molecular mechanisms of mutant C9orf72 human iPSC-derived astrocyte-mediated motor neuron deficits
Funder: Medical Research Council

Abstract: My proposal aims to use C9orf72 ALS patient-derived induced pluripotent stem cell lines to investigate the consequences of the C9orf72 mutation on human astrocyte behaviour in ALS and, specifically its impact on motor neuron (MN) function. Building on strong pilot data that shows mutant astrocyte-dependent reduction in MN neurite length, my objectives are to: 1. Elicit whether the above phenotype is contact-mediated or due to the release of astrocyte-derived soluble factors by testing the impact of conditioned media from mutant astrocytes on MNs. 2. Elucidate underlying molecular mechanisms through a combination of unbiased transcriptomic and mass spectrometric analyses. This will be followed by manipulation of the identified molecular pathways to reverse the phenotypic deficit. Methodology - The approaches I will adopt in my programme of work are all routine in my host laboratory and include; (i) Generation of enriched human astrocyte and motor neuron cultures from 3 independent ALS patient mutant C9orf72 lines. Crucially each of these has a paired isogenic gene control generated by CRISPR/Cas9. All of these lines were derived, validated and are routinely cultured in my host lab. Using these lines, I have already generated pilot data showing mutant astrocyte-mediated reduction of MN neurite length. I will extend these findings using co-cultures and quantitative immunostaining and then use mass spectrometry and / or RNA sequencing to identify the underlying mechanism of astrocyte-mediated toxicity on MNs.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: NEUROLOGICAL
Title: The AAGGG repeat expansion in RFC1 associated with late-onset ataxia and sensory neuropathy: from genetic cause to defining the functional mechanism
Funder: Medical Research Council

Abstract: Late-onset ataxia is a common reason for neurological consultation, but its cause often remains idiopathic. Ataxia primarily results from cerebellar dysfunction but can also be caused by disorders affecting the large-fibre sensory neurons (sensory neuronopathy) or the vestibular system. When in combination, this more severe late onset ataxia is termed cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS). We identified a biallelic intronic AAGGG repeat expansion in the replication factor C subunit 1 (RFC1) as a common cause of CANVAS and late-onset ataxia. The AAGGG repeat expansion does not lead to overt loss of function of RFC1, which is unexpected given the recessive pattern of inheritance of the disease and suggests that novel disease-causing mechanisms could be involved. Main objective of this project is to investigate the molecular mechanisms underlying neurodegeneration in the presence of biallelic AAGGG expansions in RFC1. We will use a combinational approach by taking advantage in vitro and Drosophila models as well as patients'-derived cell lines and tissues in order to test the presence of a dose-dependent gain-of-function of toxic pentapeptide repeat proteins encoded by the transcribed intronic repeated sequence and/or unconventional loss-of-function of tissue specific RFC1 isoforms, non-coding transcripts, neighbouring and distant genes and/or reorganization of topologically associated domains and 3D chromatin structure.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: NEUROLOGICAL
Title: Rescuing neurotrophic signalling through Rab10: a novel therapeutic target for Alzheimer's disease.
Funder: Medical Research Council

Abstract: AD is the most prevalent neurodegenerative disease and the first cause of dementia. While significant efforts have been devoted to understand its multiple genetic causes and pathological mechanisms, no current treatment have been able to stop or reverse disease progression. Several aspects of the pathophysiology of AD are regulated by neurotrophins, including production of Abeta, phosphorylation of Tau, synaptic plasticity, dendritic branching and intracellular transport; suggesting that neurotrophins play a central role in the pathological cascade. Since neurotrophic factors are secreted by diverse neuronal targets, retrograde axonal transport of signalling complexes is crucial for neurons, as exemplified by the involvement of axonal transport in several neurodegenerative diseases. Our preliminary results indicate that the GTPase Rab10 plays a key role in retrograde transport of TrkB receptors in neurons. Interestingly, recent evidence shows that phosphorylated Rab10 co-localise with Tau aggregates and around the site of senile plaques in humans. Moreover, a rare single nucleotide polymorphism of Rab10 confers protection to ApoE epsilon4 allele carriers. In this proposal we will test the hypothesis that modulation of Rab10 can overcome trafficking abnormalities in AD, restoring neurotrophic signalling and function. We propose an integrated approach to manipulate Rab10 both in vitro and in vivo, gaining a better insight into AD pathomechanisms and the therapeutic potential of modulating Rab10 function. To make our pre-clinical research program more translatable, we will explore the potential of the anti-diabetic drug metformin, a well-established regulator of Rab10-positive organelles, to regulate neurotrophic signalling in neurons in vitro and in vivo. This project is expected to further our understanding on the mechanisms of neurotrophic dysfunction in AD and test a novel therapeutic target for AD amenable to drug discovery and repositioning.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: NEUROLOGICAL
Title: Calcium-permeable AMPA receptors and their auxiliary subunits: pharmacological and molecular intervention in health and disease
Funder: Medical Research Council

Abstract: AMPARs mediate fast excitatory transmission throughout the brain, and their dynamic regulation is essential in the expression of synaptic plasticity. The calcium permeable AMPAR subtype plays an important role in the expression of normal synaptic plasticity. In addition, its increased expression or activation is implicated in several serious neurological conditions and deleterious forms of plasticity. Our programme aims to understand the molecular mechanisms, particularly the role of auxiliary subunits, in normal regulation of CP-AMPARs, and to examine their potential for manipulation in damaging forms of synaptic plasticity. We will use molecular biology, patch-clamp recording, structural biology and in vitro and in vivo pharmacology to address the following. First, we will use a novel intracellular NASPM-based technique to accurately monitor CP-AMPAR prevalence in living neurons. Parallel experiments will use a powerful fluorescent labelling approach to track the precise location of known TARP subtypes in the membrane of living cells. Second, we aim to elucidate how the newly discovered TARP-selective drugs alter AMPAR channel function and examine their utility as tools to dissect AMPAR-TARP interactions. Third, we will use newly developed structural and functional methods to identify auxiliary subunit C-tail residues critical in regulating CP-AMPAR plasticity. Fourth, we will examine auxiliary subunits mechanisms that drive CP-AMPAR plasticity under normal and deleterious conditions, and use novel TARP-selective drugs to intervene in these processes. Specifically, we will examine the identity of the auxiliary subunits underlying CP-AMPARs plasticity in ischemic damage, and the auxiliary subunits that drive CP-AMPAR plasticity in hyperalgesia, and the influence of TARP-selective drugs. We expect to obtain insight into the regulatory roles of auxiliary proteins, and reveal targets with translational value in enabling selective manipulation of CP-AMPARs.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: NEUROLOGICAL
Title: Pharmaco-circuitry of neurosteroids - regulators of mood and excitability disorders
Funder: Medical Research Council

Abstract: We will examine inhibitory transmission and neural circuit activity, focussing on GABA-ARs and their specific modulation by neurosteroids in the amygdala, in the context of mental health disorders. Our aim is to understand how dysfunctional mechanisms involving GABA inhibition and GABA-AR modulation can precipitate mental illness by studying an area of the brain that is critical for emotional processing. We will use genetic, cellular, molecular and behavioural methods to examine the interplay between neurosteroids and GABA-ARs in the amygdala. Genetic engineering will be used to selectively remove neurosteroid modulation from alpha3- and alpha5-GABA-ARs in specific amygdala cell types. Whole-cell patch clamp recording will assess synaptic and tonic GABA inhibition and their effects on excitability using native receptors in acute brain slice preparations before proceeding to whole animals to assess neural circuitry and EEG activity. Key aspects of GABA channel function, including spontaneous gating and the effects of phosphorylation will also be assessed. Application of molecular biological, biochemical and electrophysiological methods will allow us to scrutinise receptor post-translational modifications, trafficking and function. Behavioural experiments will ascertain the importance of neurosteroid regulation of amygdala GABA-ARs for anxiety phenotypes, fear processing, and cognition, for which operators will be blinded for genotype. In parallel, we will use our high-resolution structural approaches (x-ray crystallography, cryo-EM), in collaboration with our pharmaceutical partners, to examine the binding of endogenous and synthetic neurosteroids to GABA-ARs, with the aim of developing new GABAAR-specific neurosteroids as novel therapeutics. Overall, our programme aims to improve the understanding of how dysfunctional inhibition within the brain impacts on mental health and to provide new approaches for the treatment of specific disorders.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: NEUROLOGICAL
Title: Virtual presynaptic nerve terminal: a computational tool for studying synaptic transmitter release in health and disease
Funder: Medical Research Council

Abstract: The principal difficulty in studying the regulation of transmitter release is that the majority of presynaptic boutons in the brain are small (~1 micron, comparable to the diffraction limit of conventional optical microscopy ~300 nm). Data-constrained realistic computational models of presynaptic terminals are therefore essential tools that bypass the limitations of experimental approaches. Here we propose to develop a unified framework to model presynaptic terminal function, which will allow neuroscientists to explore the mechanisms of Ca2+-driven transmitter release that cannot be directly determined experimentally. We will incorporate in the model the following key functional presynaptic elements: (i) the morphological structure of a presynaptic terminal (3D geometry, distributions of voltage-gated calcium channels (VGCCs) and release ready synaptic vesicles (SVs)); (ii) pre-defined VGCC kinetic models; (iii) Ca2+ diffusion, buffering and extrusion mechanisms; (iv) intracellular Ca2+ stores (v) Ca2+ activation of SV exocytosis and replenishment. During the project, we will collaborate with a group of world-leading laboratories specialising in synaptic physiology. This will allow us to develop experimentally constrained models of several canonical presynaptic terminals and to investigate how the complex interplay among distinct functional presynaptic components regulates transmitter release and use-dependent plasticity in different synapses. We will also use the model to investigate in silico the mechanisms of presynaptic channelopathies, with a particular focus on some forms of migraine, epilepsy and ataxias caused by mutations in presynaptic Cav2.1 (P/Q-type) VGCCs and Kv1.1 potassium channels. We anticipate that the release of the unified presynaptic model to the public domain will provide a powerful, currently unavailable resource, which will facilitate experimental research in the field of synaptic physiology.


Research activity codes: 1.4 METHODOLOGIES AND MEASUREMENTS
Health categories: NEUROLOGICAL
Title: Regulation of 3D genome organisation and function in axonal regeneration
Funder: Medical Research Council

Abstract: Following injury, peripheral nervous system axons regenerate while axons in the central nervous system fail to do so. The dorsal root ganglia (DRG) bipolar neurons allow investigating the molecular mechanisms of this differential regenerative ability: a single cell body extends one regenerative competent axon in the peripheral nerves and one regenerative incompetent branch into the spinal cord. We recently performed genome-wide gene expression and epigenetic studies (ATAC and ChIPSeq for histone PTMs) in the mouse DRG after peripheral nerve injury (SNA) vs central spinal dorsal column axotomy (DCA). These revealed that chromatin accessibility is modified via histone acetylation affecting the transcription programme facilitating regeneration following SNA but not after DCA. We also discovered enrichment in CCCTC binding factor (CTCF)-dependent transcriptional binding sites at promoters of regenerative genes in proximity of more accessible chromatin and that conditional neuronal deletion of CTCF leads to impaired axonal regeneration after sciatic nerve injury. Since CTCF plays an important role in 3D genome organisation, here we hypothesize that CTCF-mediated genome organization contributes to the injury-induced transcriptional programme and to axonal regeneration. We will investigate (i) CTCF-dependent and independent mechanisms of chromatin and transcriptional regulation following axonal injury by Circular Chromosome Conformation Capture sequencing (4Cseq), RNAseq and CTCF ChIPseq from DRG after spinal or sciatic injury (DCA vs SNA) comparing control versus CTCF-deleted DRG neurons; (ii) CTCF post-translational modifications following sciatic versus spinal injury that affect CTCF activity and DRG regenerative growth; (iii) the ability to enhance axonal regeneration following injury, by altering 3D genome conformation, via CRISPR-cas9 genome editing. In summary, these studies will provide a novel understanding of chromatin-dependent regulation of axonal regeneration.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: NEUROLOGICAL
Title: Modelling dementia progression based on machine learning and simulations
Funder: Medical Research Council

Abstract: Our basic strategy is to 1) develop machine learning and dynamic model with available public datasets (ADNI, DPUK, Newcastle data) and 2) validate them with the against other cohorts (UK Biobank and Korea data). In this project, we will combine neuroimaging dataset including DWI, resting-sate fMRI, and PET data. DEVELOPING MACHINE LEARNING APPROACHES Using structural connectivity as measured with diffusion MRI in prodromal dementia, starting with connectivity in healthy subjects, and using a computer simulation to study the progression over time towards healthy ageing or dementia, we found that pathophysiological alterations associated with dementia become significantly apparent before the onset of symptoms, meeting diagnostic criteria for clinical dementia, indicating a potential biomarker for progression towards dementia. This machine learning approach could be improved further by using deep learning. Indeed, Korea University, in addition to providing further training and test datasets, has already developed a deep learning approach for dementia brain connectivity data. We will extend these approaches to develop a model of disease progression, looking at changes in white matter and gray matter organization, and testing the role of different underlying biological mechanisms through computational modelling. VALIDATING OUR APPROACH IN A CLINICAL SETTING This study utilizes existing datasets for training our approach. We use severable studies to ensure that predicting disease progression is reliable across study sites (UK vs. Korea) and patient cohorts. Note that studies, in addition to neuroimaging data, include the complete set of cognitive and clinical scores (MMSE, CAMCOG, UPDRS-III, NPI-Hall, CAF, Cornell-DS) as well as medication information. In a second step, we will use other datasets (UK Biobank and data from Korea University) as test datasets to see whether our approach can deal with novel datasets.


Research activity codes: 2.5 RESEARCH DESIGN AND METHODOLOGIES (AETIOLOGY)
Health categories: NEUROLOGICAL
Title: Restoring hippocampal-cortical circuit and memory dysfunction in prodromal Alzheimer's Disease.
Funder: Medical Research Council

Abstract: Cortical and hippocampal neurons exist as two broad subtypes, excitatory glutamatergic neurons and inhibitory GABAergic neurons. Recent years have seen a huge leap in our understanding of their relative contribution to normal brain function. To address our project aims, we will employ two main experimental paradigms: (1) electrophysiology recordings in acute brain slices and in vivo of hippocampus and retrosplenial cortex and (2) behavioural assays. We will probe the role of somatostatin positive inhibitory interneurons (SST+) in neuronal oscillations both in control mice and mouse models of Alzheimer's Disease (AD, either through application of Abeta oligomers or in AppNL-G-F transgenic mice). We will use SST-Cre mice to deliver opsins to SST+ neurons only. Part of the project will require the generation of SST-Cre x AppNL-G-F mice to test the rescuing effect of SST+ stimulation in AD mice performing RSC-dependent memory tasks that are sensitive to prodromal AD dysfunction. A final set of experiments aims to explore possible links between the ameliorative effect of sensory stimulation on AD-associated deficits to the rescuing effect of SST+ stimulation.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES;5.2 CELLULAR AND GENE THERAPIES
Health categories: NEUROLOGICAL
Title: Ultra-high resolution 7T MRI mapping of basal ganglia connectivity on an individual patient basis for Paediatric Deep Brain Stimulation (DBS)
Funder: Medical Research Council

Abstract: Dystonia is a painful disabling neurological condition characterised by involuntary muscle contractions. The commonest cause of dystonia in childhood is Cerebral Palsy (CP), affecting 2-3:1000 live births in the UK. Pharmacological treatments for dystonia in CP are often ineffective, and frequently produce significant side effects. This has led to increasing interest in Deep Brain Stimulation (DBS), which involves the neurosurgical placement of electrodes into one of the nuclei of the basal ganglia. Generally positive results are seen following DBS in children with CP, but the degree of improvement varies between children for reasons which are currently unclear. The success of DBS depends upon the accurate placement of stimulating electrodes. In children with CP, the boundaries of the target nuclei for DBS may be difficult to delineate on clinically available MRI scanners (1.5/3T field strength). The basal ganglia nuclei are known to be internally sub-divided into functional territories, the motor region being the target for DBS. Whilst the motor region cannot be visualised on structural MRI sequences, advanced imaging techniques can be used to define the region on the basis of measures of functional connectivity (resting state fMRI analysis) or structural connectivity (tractography analysis). Connectivity analysis can also be used to inform us of the distributed brain network through which implanted electrodes act, improving our understanding of the mechanisms of action of DBS. We hypothesise that the improved signal to noise ratio offered by ultra-high resolution 7T MRI will enable us to more accurately image the basal ganglia and its connections, and to map the brain network accessed by implanted DBS electrodes. We will perform a comparative study for a cohort of children undergoing DBS surgery, obtaining scans on both 3T and 7T MRI scanners. Following surgery, DBS Evoked Potentials will be used to help us begin to validate the results of connectivity analysis.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;4.3 INFLUENCES AND IMPACT
Health categories: NEUROLOGICAL
Title: Novel MRI Biomarkers in Neuromuscular Disease
Funder: Medical Research Council

Abstract: There is a pressing need to develop sensitive and reproducible biomarkers of motor unit loss in motor neuron disease (MND). We recently developed a novel diffusion-weighted MRI protocol (Motor Unit MRI or MUMRI) sensitive to the spontaneous or electrically-evoked contraction of individual skeletal motor units (Ann Neurol 2019 Mar;85(3):455-459). In this fellowship I will a) validate this novel imaging technique against current clinical measures of motor unit fasciculation (ultrasound and surface electromyography), b) perform a longitudinal study of motor unit dysfunction and degeneration in the lower limbs of patients with MND, using a combination of MUMRI and conventional imaging modalities (T1, STIR, T2 mapping, and 3-point Dixon fat fraction) and c) develop a 'whole-body' MUMRI imaging system with which to investigate the spread of fasciculation both within and between skeletal muscles. The outcomes of this project will be a validated and trial-ready MR-based skeletal muscle imaging system suitable for use in both clinical diagnosis and longitudinal studies of novel therapies. The pattern whereby fasciculation and structural changes develop in muscles within and between different body regions will also inform the ongoing debate as to whether or not MND develops by prion-like spread of aberrantly folded proteins between neighbouring populations of motor neurons or as an inherently diffuse process.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: NEUROLOGICAL
Title: Brain Iron Toxicity and Neurodegeneration - MRI study at 7T
Funder: Medical Research Council

Abstract: Ultra-High Field MRI can be used to study variation in sub-structural markers of neurodegeneration with time and has the potential to identify and possibly quantify another pathological element in Alzheimer's disease, by using a non-invasive and cheaper method than Amyloid/tau examination. Changes in brain volume can be monitored with great sensitivity, even in very small regions such as the hippocampal subfields. The recently developed MRI technique of quantitative susceptibility mapping (QSM) can quantify the concentration of paramagnetic materials including iron, with greatly increased sensitivity at 7T. The use of QSM will allow the iron content in substructures and strategically important areas of the brain to be characterised over time in patients with Alzheimer's disease. Knowledge of the distribution of iron deposition in brain tissue of patients with Alzheimer's disease can, characterise the heterogeneity of Alzheimer's pathology, and help us to understand the relevance of iron in neurodegeneration and its clinical and cognitive implications. Characterisation of the trajectory of brain tissue iron content in the Alzheimer's group will improve our knowledge of the relationship between iron accumulation and local neurodegeneration/hippocampal volume loss, in relation to Amyloid/tau-pathology and APO status. This project may potentially identify an imaging-based biomarker which can either provide a disease-specific pathological marker (i.e. presence of iron in hippocampal subfields) or a pre-neurodegeneration indicator in patients with Alzheimer's disease. In follow-on work, we would test whether any imaging biomarker found in 7T MRI could also be exploited at 3T.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: NEUROLOGICAL