GW4 BioMed2 MRC DTP Studentship: Epigenetic regulation of microglial gene expression in Alzheimer's disease

Closing Date
26 Nov 2021
Salary
tuition fees funded, doctoral stipend £15,609 per annum
Address
Cardiff University

Neuroinflammation is emerging as a key pathogenetic event in risk and progression of Alzheimer’s disease (AD). Genome wide association studies have identified single nucleotide polymorphisms (SNPs) at genetic loci associated with microglia function enriched in AD patient cohorts, however precisely 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. To better understand these events, we will profile the epigenome of human microglia in an in vitro cell culture model 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 human induced pluripotent stem cell (hiPSC) model of amyloid accumulation will be utilised. KOLF2 hiPSCs will be differentiated to the following microglia/mature cortical neuron co-cultures: 1) wild type neurons, wild type microglia (WT co-culture); 2) APPSWE/IND expressing neurons, wild microglia (AD co-culture). Microglia will next be enriched from the co-cultures using CD11 magnetic beads (MACS) for cell separation and genomic DNA and RNA isolation.    1b. UEx: To define 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 RNASeq transcriptome profiling. Epigenomic and transcriptomic profiles will be bioinformatically analysed using established analysis pipelines and bioinformatic approaches. From these data, prioritised genes 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 qPCR and/or antibody staining, assessing microglia in either WT or APP co-cultures. From this we will triage 3 genes where methylation is associated with robust changes in expression for further analysis. We will next optimise and validate mis-expression of the three lead genes in hiPSC microglia, utilising CRISPRi for gene disruption (Cas9-KREB, CLYBL safe harbour integration) or CRISPRa (dCas9-VP64) for activation (transgenic siRNA/cDNA as contingency). Altered gene expression will be confirmed by qPCR, western blotting and immunostaining.    2b. CU: AD-relevant microglia phenotypes will be assessed in microglia mis-expressing the lead candidate genes. 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 (Opera Phenix).   Our approach will define AD relevant epigenetic regulation of microglia and explore how altered expression of these genes contributes to neuroinflammatory function.  

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. However, the DTP also welcomes students from non-medical backgrounds, especially in areas of computing, mathematics and 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. This will be at least 6.5 in IELTS or an acceptable equivalent. 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 'offer to study' to your chosen institution(s):

To submit a formal application via Cardiff University’s online application service, click the 'Institution Website' button on this advert; in the ‘Apply’ box at the top-right of the page, select Qualification (Doctor of Philosophy), Mode of Study (Full Time) and Start Date (October 2022). This will take you to the application portal.

Candidates must submit the following:

 • Supporting statement

 • CV

 • Qualification certificates

 • Proof of English language (if applicable) 

In the research proposal section of the application, specify the project title and supervisors of the project. In the funding section, select “I will be applying for a scholarship/grant” and specify advertised funding from GW4 BioMed2 MRC. If you are applying for more than one Cardiff University project, please note this in the research proposal section as the form only allows you to enter one title.

Please complete the online application form by 5.00pm on 26th November 2021. If you are shortlisted for interview, you will be notified by 28th January 2022. Interviews will be held virtually on 16th and 17th February 2022.

Further Information

For informal enquiries, please contact GW4BioMed@cardiff.ac.uk

For project related queries, please contact the respective supervisors listed on the projects.