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Modelling neurodegenerative diseases in human iPSC-derived neurons

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cam.depositDate2022-05-16
cam.restrictionthesis_access_embargoed
cam.supervisorRubinsztein, David
cam.supervisorFischbeck, Kenneth
cam.supervisor.orcidRubinsztein, David [0000-0001-5002-5263]
dc.contributor.authorPrestil, Ryan
dc.date.accessioned2022-05-17T15:56:55Z
dc.date.available2022-05-17T15:56:55Z
dc.date.submitted2021-07-28
dc.date.updated2022-05-16T12:14:25Z
dc.description.abstractNeurodegeneration is a pathology shared by a varied class of diseases, and many of the mutations that are known to cause such diseases have been linked to protein aggregation and autophagy dysfunction. Improvements to gene editing and neuronal differentiation strategies have enabled the derivation of in vitro disease models using human iPSC-derived neurons to provide a more accurate understanding of how disease mutations affect neuronal health. I first sought to model the polyglutamine disease spinal and bulbar muscular atrophy (SBMA), detailed in Chapter 4. Using iPSCs derived from a healthy donor and an SBMA patient, the CAG repeat of the endogenous androgen receptor (AR) gene was CRISPR-edited to encode a series of lengths or an early stop codon. However, AR expression was silenced upon transcription factor-mediated differentiation to a lower motor neuron-like state, and chemical differentiation prevented ligand-induced AR nuclear translocation. Deriving the cell lines in this work highlighted that purification of transgenic cells is a key bottleneck to gene editing. I therefore adapted a synthetic marker gene that presents a streptavidin binding peptide (SBP) tag on the extracellular membrane, detailed in Chapter 3. Expression of this tag in iPSCs enabled transient fluorophore staining and effective sorting of mixed populations with magnetic streptavidin beads. Finally, Chapter 5 establishes L1CAM as a novel autophagy modulator; iPSC-derived neurons showed that reduction of the L1CAM transcript with shRNAs, but not genetic knockout of the L1CAM protein, is sufficient to reduce transcription of the ATG8 gene family, which are core components of macroautophagy. This work exemplified both the strengths and weaknesses of iPSC-derived neurons; namely, they are tractable and able to recapitulate neural phenotypes, but deriving new model lines requires a high initial investment, so adequate proof-of-concept is crucial.
dc.description.sponsorshipNational Institutes of Health Oxford-Cambridge Scholars Program National Institute of Neurological Disorders and Stroke Cambridge International Trust
dc.identifier.doi10.17863/CAM.84662
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/337246
dc.language.isoeng
dc.publisher.institutionUniversity of Cambridge
dc.rightsAll Rights Reserved
dc.rights.urihttps://www.rioxx.net/licenses/all-rights-reserved/
dc.subjectneurodegeneration
dc.subjectautophagy
dc.subjectstem cell
dc.subjectdisease modelling
dc.subjectSpinal and bulbar muscular atrophy
dc.subjectcell sorting
dc.subjectL1CAM
dc.titleModelling neurodegenerative diseases in human iPSC-derived neurons
dc.typeThesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnameDoctor of Philosophy (PhD)
pubs.licence-display-nameApollo Repository Deposit Licence Agreement
pubs.licence-identifierapollo-deposit-licence-2-1
rioxxterms.licenseref.urihttps://www.rioxx.net/licenses/all-rights-reserved/
rioxxterms.typeThesis

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