Neuroinflammation is emerging as a key pathogenetic event in risk and progression of Alzheimer’s disease (AD). GWAS have identified single nucleotide polymorphisms (SNPs) at genetic loci associated with microglia function enriched in AD cohorts, however how these alter gene expression within loci is often unclear. Profiling transcriptional regulation of AD associated gene by epigenome wide association studies of post-mortem human cortex has identified enriched patterns of DNA methylation in microglial genes of AD patients. Whilst further illuminating the contribution of microglia in AD pathogenesis, a caveat of these experiments is the tissue utilised; post-mortem samples of mixed cell types derived from patients dying with late-stage AD with profound neurodegeneration and inflammation. Such tissues may not capture more subtle, early changes in epigenomic regulation that underpin functional changes in cells in prodromal patients. To better understand these events, we will profile the epigenome of human microglia in in vitro co-culture models of AD, hypothesising that essential microglial functions are disrupted through altered epigenetic regulation of microglia specific genes in AD.
Aim 1: To define epigenetic regulation of microglia genes associated with extracellular amyloid 1a. CU: To generate an AD relevant population of microglia for epigenomic profiling, a hiPSC model of amyloid accumulation will be utilised. APP mutant and isogenic control KOLF2 hiPSCs will be differentiated to establish the following microglia/cortical neuron co-cultures: 1) wild type neurons, wild type microglia (WT co-culture); 2) APP mutant expressing neurons, wild microglia (AD co-culture). Microglia will next be recovered from the co-cultures using CD11 magnetic beads (MACS) for cell separation and genomic DNA and RNA isolation. 1b. UEx: To define the genome wide patterns of DNA methylation and gene regulation, microglia DNA and RNA collected from WT or AD co-cultures will undergo quantitative genome wide profiling for methylation by microarray profiling (Infinium MethylationEPIC BeadChip platform, 850k CpG sites) and bulk RNASeq transcriptome profiling. Epigenomic and transcriptomic profiles will be bioinformatically analysed using established analysis and bioinformatic approaches. Differentially regulated targets will be triaged for further analysis, based on i) statistical significance, ii) association with the expression of proximal genes, iii) contribution to microglia specific function and iv) potential for therapeutic intervention.
Aim 2: Contribution of epigenetically regulated microglia genes in AD-relevant phenotypes. 2a. CU/UEx: Methylation associated regulation of genes in prioritised loci will be further validated by bisulphite sequencing, qPCR and/or antibody staining, assessing microglia in either WT or APP co-cultures. We will triage 3 genes where methylation is associated with robust changes in expression for further analysis. We will next mis-express three lead genes in hiPSC microglia, where appropriate utilising targeted methylation by Cas9-DNMT, and/or CRISPRi for gene disruption (Cas9- KREB), CRISPRa (dCas9-VP64) for activation (transgenic siRNA/cDNA as contingency). Changes in gene expression will be confirmed by qPCR, western blotting and immunostaining. 2b. CU: The consequences of gene manipulation in 2a will be examined for AD-relevant microglia phenotypes. Assays will include inflammatory responses of microglia stimulated with INFΥ/LPS, quantified by cytokine release via a flow cytometry-based immunoassay (BD cytokine bead array); changes in morphology and motility of INFΥ/LPS stimulated microglia in neuronal WT and AD co-cultures; and phagocytosis of pHrodo labelled E. coli or fluorescent amyloid oligomers assessed by live imaging. Our approach will define AD relevant epigenetic regulation of microglia and explore how altered expression of these genes contributes to neuroinflammatory function.
Applications open on 2nd September 2022 and close at 5.00pm on 2nd November 2022.
About the GW4 BioMed2 Doctoral Training Partnership
The partnership brings together the Universities of Bath, Bristol, Cardiff (lead) and Exeter to develop the next generation of biomedical researchers. Students will have access to the combined research strengths, training expertise and resources of the four research-intensive universities, with opportunities to participate in interdisciplinary and 'team science'. The DTP already has over 90 studentships over 6 cohorts in its first phase., along with 20 students in its second phase.
Eligibility
Residency: The GW4 BioMed2 MRC DTP studentships are available to UK and International applicants. Following Brexit, the UKRI now classifies EU students as international unless they have rights under the EU Settlement Scheme. The GW4 partners have all agreed to cover the difference in costs between home and international tuition fees. This means that international candidates will not be expected to cover this cost and will be fully funded but need to be aware that they will be required to cover the cost of their student visa, healthcare surcharge and other costs of moving to the UK to do a PhD. All studentships will be competitively awarded and there is a limit to the number of International students that we can accept into our programme (up to 30% cap across our partners per annum).
Academic criteria: Applicants for a studentship must have obtained, or be about to obtain, a UK degree, or the equivalent qualification gained outside the UK, in an appropriate area of medical sciences, computing, mathematics or the physical sciences. Please check the entry requirements of the home institution for each project of interest before completing an application. Academic qualifications are considered alongside significant relevant non-academic experience.
English requirements: If English is not your first language you will need to meet the English language requirements of the university that will host your PhD by the start of the programme. Please refer to the relevant university for further information.
How to Apply
A list of all the projects and how to apply is available on our website at gw4biomed.ac.uk. You may apply for up to 2 projects.
Please complete an application to the GW4 BioMed2 MRC DTP for an ‘offer of funding’. You may also need to make an application for an 'offer to study' to your chosen institution(s) – further details are on the website.
Please complete the online application form by 5.00pm on Wednesday, 2nd November 2022. If you are shortlisted for interview, you will be notified by Friday 16th December 2022. Interviews will be held virtually on 25th and 26th January 2023.
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