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DiMeN Doctoral Training Partnership: Characterizing the functions of a microcephaly-related gene in a knock-out mouse model using in vivo MRI, histology and cellular/molecular biology approaches

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  • Full or part time
    Prof H Poptani
    Dr A Plagge
  • Application Deadline
    No more applications being accepted
  • Competition Funded PhD Project (European/UK Students Only)
    Competition Funded PhD Project (European/UK Students Only)

Project Description

Characterizing the functions of a microcephaly-related gene in a knock-out mouse model using in vivo MRI, histology and cellular/molecular biology approaches.

This will be an interdisciplinary PhD project, which investigates a knock-out (KO) mouse model for a human neurodevelopmental disorder. It provides an opportunity to learn a variety of techniques and methods through application of neuroimaging (magnetic resonance imaging, MRI), histological analyses of brain abnormalities, primary cultures of neural progenitor cells (NPCs) and neurons, as well as molecular genetic analyses of gene regulation.

Rare human genetic diseases can highlight previously unrecognised, but important gene functions. Mutations in the human TRAPPC9 gene cause microcephaly, intellectual disability, speech impairment and developmental delays. The Trappc9 protein functions in the regulation of intracellular transport and vesicle size. Using a novel Trappc9 KO mouse line, we have confirmed microcephaly in young adult mutant mice by measuring brain weights as well as brain volumes via high-resolution MRI. μMRI also indicates the specific brain regions affected in KO brains. We have characterised the Trappc9 expression pattern in a subset of neurons and NPCs of the brain and in neurosphere cultures. Furthermore, we found that the Trappc9 gene is regulated by epigenetic mechanisms (genomic imprinting) in the mouse brain. Transcription occurs preferentially from the maternally inherited allele, and this is accompanied by tissue-specific differences in DNA methylation.

In this project we intend to further characterize the disease symptoms/brain phenotypes of the knock-out mouse model, determine the cellular functions of the Trappc9 protein in neurons and progenitor cells and shed light on the epigenetic regulation of the gene.
Specifically, we want to apply more sophisticated MRI techniques (diffusion imaging methods) to determine micro-structural changes in brain sub-regions and neurite density. Histological techniques will be used to validate findings. Since the developmental onset of microcephaly is currently unclear, we intend to undertake a longitudinal in vivo MRI study to examine total brain volumes at various postnatal and juvenile stages in a cohort of mice.
Complementary to the analysis of brain abnormalities, we want to functionally assess neural progenitor cells in vivo and ex vivo. We will label proliferating NPCs in vivo via BrdU incorporation and characterise differences between wild-type and KO mice in combination with immunohistochemistry for stem cell markers and apoptosis. Ex vivo, we will assess NPC proliferation and differentiation using neurosphere cultures. Primary neuron cultures can be grown in microfluidic chambers to evaluate retrograde transport of signalling endosomes using specific fluorescent endosome-uptake markers and live-cell imaging.
A third aspect is concerned with the epigenetic regulation of murine Trappc9 in comparison to the human gene regulation. We are preparing ChIP-assays for histone modifications and chromosome conformation capture experiments for promoter-enhancer interactions to verify the functions of DNA-binding proteins and allele-specific DNA methylation.

By the end of this programme, the student will have acquired a wide range of molecular and cellular biology, imaging, in vivo physiology and quantitative skills, which will provide a good basis for a further step in biomedical research. Apart from training in the laboratories of the primary and secondary supervisors, the student will gain additional skills through participation in training events organised for all students of this MRC Doctoral Training Partnership scheme.

Laboratory Prof H. Poptani: https://www.liverpool.ac.uk/translational-medicine/staff/harish-poptani/
Laboratory Dr A. Plagge: https://www.liverpool.ac.uk/translational-medicine/staff/antonius-plagge/
University of Liverpool Centre for Preclinical Imaging: https://www.liverpool.ac.uk/translational-medicine/research/centre-for-preclinical-imaging/

Project background literature:
Mochida GH, et al.; A Truncating Mutation of TRAPPC9 Is Associated with Autosomal-Recessive Intellectual Disability and Postnatal Microcephaly. Am J Hum Genet. 2009, 85:897-902. doi: 10.1016/j.ajhg.2009.10.027
Li C, et al.; COPI-TRAPPII activates Rab18 and regulates its lipid droplet association. EMBO J. 2017, 36:441-457. doi: 10.15252/embj.201694866

Funding Notes

This studentship is part of the MRC Discovery Medicine North (DiMeN) partnership and is funded for 3.5 years. Including the following financial support:
Tax-free maintenance grant at the national UK Research Council rate
Full payment of tuition fees at the standard UK/EU rate
Research training support grant (RTSG)
Travel allowance for attendance at UK and international meetings
Opportunity to apply for Flexible Funds for further training and development
Please carefully read eligibility requirements and how to apply on our website, then use the link on this page to submit an application: http://www.dimen.org.uk/how-to-apply/application-overview

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