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PTEN as a Therapeutic Target for the Promotion of Cell Survival in Cell Models of Amyotrophic Lateral Sclerosis

Ashman, Cassy J (2017) PTEN as a Therapeutic Target for the Promotion of Cell Survival in Cell Models of Amyotrophic Lateral Sclerosis. PhD thesis, University of Sheffield.

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Abstract

Rationale & Hypothesis: ALS is a fatal neurodegenerative disorder for which a GGGGCC hexanucleotide repeat expansion in C9ORF72 has been identified to be the most common genetic cause. Pathologically, ALS is characterised by premature death of motor neurons. The inhibition of Phosphatase and Tensin Homologue Deleted on Chromosome Ten (PTEN) in the PI3K cell survival cascade has been found to promote cell survival, and a newly described drug Scriptaid has been found to offer neuroprotection through PTEN inhibition in this pathway. We hypothesised that inhibiting PTEN in cell models of ALS could improve cell survival. Methodology and Objectives: For this investigation, we aimed to generate and characterise induced pluripotent stem (iPS) cell-derived motor neurons as in vitro models of ALS, from C9ORF72-ALS patient and sex/age matched healthy control primary fibroblast cells. Our objectives were to determine how PTEN manipulation through inhibition with Scriptaid or knockdown with short hairpin RNA (shRNA) lentiviral particles affects cell number and survival, comparing the newly generated iPS-derived cell models to established cell models of ALS including Neuroblastoma spinal cord (NSC)-34 cells and the primary fibroblast cells the iPS cells were derived from. Findings: We were able to successfully differentiate iPS cells into neurons and motor neurons of intermediate maturity in both patient and control cells, with C9ORF72 patient cells maintaining hallmarks of the repeat expansion. Results revealed fibroblast cells from C9ORF72 cases exhibited significantly lower levels of basal PTEN compared to control cells, however after the conversion to iPS-derived motor neurons, no significant difference between patient or control levels of PTEN expression were identified. PTEN knockdown in iPS-derived cells was found to cause a concomitant increase in PI3K pathway activation, and revealed a significant protection of cell number for up to three weeks in patient cells compared to controls after manipulation. However, PTEN inhibition through the use of Scriptaid had no significant effect on activating this cell survival pathway or promoting motor neuron survival in our investigations. Conclusions and Future Avenues: Together, these results highlight that PTEN inhibition through genetic manipulation activates the PI3K cell survival pathway to promote a positive therapeutic effect in ALS iPS-derived cell models. However, small molecule modifier of PTEN Scriptaid does not promote motor neuron cell survival or activate cell survival pathways in the cell models of ALS tested, suggesting that future experiments should use alternative methods of inhibiting PTEN directly for successful patient therapies. Comparing models, iPS cells offer a promising cell model to investigate PTEN manipulation in ALS, however further work is still needed to optimise the differentiation protocol to improve maturity and functional parameters.

Item Type: Thesis (PhD)
Academic Units: The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield)
The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield) > Medicine (Sheffield)
Identification Number/EthosID: uk.bl.ethos.715096
Depositing User: Miss Cassy J Ashman
Date Deposited: 26 Jun 2017 08:29
Last Modified: 12 Oct 2018 09:41
URI: http://etheses.whiterose.ac.uk/id/eprint/17666

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