Jestice, Lydia Helen ORCID: 0000-0002-6679-5067
(2025)
Investigating axonal transport in Charcot-Marie-Tooth disease Type 2A using a human embryonic stem cell model.
PhD thesis, University of Sheffield.
Abstract
Charcot-Marie-Tooth (CMT) disease is one of the most common forms of inherited peripheral neuropathy and has many different subtypes. One such subtype is sensory and motor neuropathy CMT Type 2A (CMT2A), for which no treatments currently exist. CMT2A is caused by mutations in Mitofusin 2 (MFN2), and it is unknown how these mutations drive disease. Hence, I set out to create the first human embryonic stem cell (hESC) model of CMT2A to investigate the impact of a CMT2A-causing mutation in a disease-relevant cell type.
I generated a panel of CMT2A hESC clones by introducing the disease-causing heterozygous R94Q mutation into Mitofusin 2 via CRISPR-Cas9 editing. The clone panel was subsequently differentiated into a disease-relevant cell type: limb-innervating motor neurons. The introduction of the CMT2A-causing mutation had no effect on differentiation. Limb-innervating motor neurons containing the disease-causing mutation displayed a mitochondrial trafficking defect characterised by a reduction in the number of motile mitochondria and the motile dynamics. The reduction in motile mitochondria could be rescued via small molecule inhibition of the deacetylase HDAC6. Furthermore, using protein over-expression and co-immunoprecipitation, I also show that MFN2 containing the R94Q mutation interacts more strongly with the trafficking cargo adapter protein TRAK1, leading to TRAK1 having a reduced interaction with the axonal motor protein kinesin.
Overall, I have successfully created hESCs containing a CMT2A-causing mutation that can be differentiated into limb-innervating neurons, providing a new in vitro platform for CMT2A research. Additionally, results here contribute to evidence that axonal transport deficits are a common CMT2 hallmark. This work provides a new hypothesis for CMT2A pathophysiology and is a foundation for the further study of axonal transport machinery and its functionality in CMT2A.
Metadata
Supervisors: | Barbaric, Ivana and De Vos, Kurt |
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Keywords: | CMT2A, Mitofusin 2, TRAK1, hESC, differentiation, axonal transport of mitochondria |
Awarding institution: | University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Science (Sheffield) > School of Biosciences (Sheffield) |
Date Deposited: | 30 Sep 2025 14:25 |
Last Modified: | 30 Sep 2025 14:25 |
Open Archives Initiative ID (OAI ID): | oai:etheses.whiterose.ac.uk:37538 |
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