MRC DiMeN Doctoral Training Partnership: Functional and genetic analyses of HSPB mutations underlying progressive neuronal deficits in Charcot-Marie-Tooth disease

Understanding the cellular and molecular mechanisms underlying progressive neurological deterioration is a key challenge in modern biomedical research. This PhD project aims to investigate the role of small heat shock proteins in Charcot-Marie Tooth disease.

Background: Charcot-Marie-Tooth (CMT) disease is one of the most commonly inherited progressive neurological disorders, characterised by muscle degeneration and neurosensory deficits and caused by mutations in neuronal proteins. Small heat shock proteins (sHSPs) are a family of ubiquitously expressed ATP-independent chaperones that regulate proteostasis through dynamic interactions with client proteins. Mutations in two human sHSPs, HSPB1 and HSPB8, are specific causative factors in CMT; however, the mechanisms by which these mutations create progressive defects are largely unknown. Expression of individual HSPB point mutations in C. elegans has confirmed CMT-like phenotypes, identifying progressive dysfunction of neurotransmitter release and evidence of neuromuscular degeneration. Preliminary tandem mass tag proteomics and bio-informatic analyses have detected novel changes in metabolic protein expression in response to HSPB mutation.

Objectives and Experimental Approach: This studentship will address the role of sHSP mutations in CMT and explore novel links to neuronal metabolism.
Aim 1: To explore and extend the identified connection between HSPB mutation and metabolism. Working in the Barclay lab, the student will use modern genetic techniques to assess functional interactions between metabolic proteins and HSPB-dependent deficits. Direct effects on metabolism will be quantified in vivo using Cas9-CRISPR gene editing or standard transgenics to create strains expressing HSPBs in tandem with genetically-encoded metabolic reporters.
Aim 2: To identify alterations to the interacting client proteome for HSPB1 and HSPB8. Under supervision of Barclay and Hammond, the student will undertake in vivo proximity-dependent biotin labelling proteomics in C. elegans. Nematodes will be genetically engineered with APEX- or miniTurbo-tagged HSPBs followed by biotin labelling, mass-spec identification and bio-informatic analyses. Positive hits will be validated and explored further by genetic approaches.
Aim 3: To examine cellular mechanisms underlying progressive neurotransmission dysfunction in response to HSPB mutation and extend to investigate novel interactions with client and metabolic proteins. Working in the Seward lab at the University of Sheffield, the student will quantify precise effects on vesicle docking and fusion using patch-clamp electrophysiology, electrochemistry and total internal reflection fluorescence microscopy.

Novelty and Timeliness: Modern genomics generates large lists of mutations linked with specific human diseases without providing sufficient information to the underlying mechanisms. This project capitalises on recent genomic advances to investigate sHSP function and CMT disease.

Student training: The student undertaking this project will receive expert training in a broad combination of modern genetic, cellular and molecular approaches combined with in vivo proteomics and bio-informatics to deliver novel insights into progressive neurological dysfunction.

Benefits of being in the DiMeN DTP:
This project is part of the Discovery Medicine North Doctoral Training Partnership (DiMeN DTP), a diverse community of PhD students across the North of England researching the major health problems facing the world today. Our partner institutions (Universities of Leeds, Liverpool, Newcastle and Sheffield) are internationally recognised as centres of research excellence and can offer you access to state-of the-art facilities to deliver high impact research.
We are very proud of our student-centred ethos and committed to supporting you throughout your PhD. As part of the DTP, we offer bespoke training in key skills sought after in early career researchers, as well as opportunities to broaden your career horizons in a range of non-academic sectors.

Being funded by the MRC means you can access additional funding for research placements, international training opportunities or internships in science policy, science communication and beyond. See how our current DiMeN students have benefited from this funding here: http://www.dimen.org.uk/overview/student-profiles/flexible-supplement-awards
Further information on the programme can be found on our website:
http://www.dimen.org.uk/