UCL Discovery
UCL home » Library Services » Electronic resources » UCL Discovery

Functional characterisation of aromatic L-amino acid decarboxylase (AADC) deficiency using a patient-derived dopaminergic cell model

Kramer, Karolin; (2019) Functional characterisation of aromatic L-amino acid decarboxylase (AADC) deficiency using a patient-derived dopaminergic cell model. Doctoral thesis (Ph.D), UCL (University College London). Green open access

[thumbnail of Krämer_10083710_Thesis.pdf]
Preview
Text
Krämer_10083710_Thesis.pdf - Accepted version

Download (7MB) | Preview

Abstract

Background: Aromatic L-amino acid decarboxylase (AADC) deficiency is a severe pharmacoresistant neurological disorder due to inherited autosomal recessive loss-of-function mutations in the DDC gene. The resultant impairment of AADC enzyme activity severely impacts on monoamine synthesis, leading to reduced levels of dopamine and serotonin. Affected patients present with marked neurodevelopmental delay, hypotonia, oculogyric crises and autonomic dysfunction. Currently, there are few truly disease-modifying therapies. Aims: To generate AADC patient-derived induced pluripotent stem cells (iPSC) for subsequent differentiation into midbrain dopaminergic (mDA) neurons, and to utilise this model to better define disease mechanisms and test novel therapeutic strategies. Methods: Patient and age-matched control fibroblasts were reprogrammed into iPSC using Sendai Virus methods. A modified dual SMAD inhibition protocol was then utilised for differentiation of all iPSC lines to day 65 of maturation. The generated neuronal model was then analysed for mature mDA neuronal identity and AADC disease-specific features. Results: iPSC lines were generated from skin fibroblasts derived from two patients with AADC deficiency. One patient harboured a homozygous missense mutation (p.R347G) and the other was a compound heterozygote for a nonsense variant (p.Arg7*) and missense mutation (p.C100S) in DDC. For the project two iPSC lines from one age-matched control subject were used that were previously reprogrammed in my host laboratory. Generated iPSC lines were confirmed as being truly pluripotent, then successfully differentiated into midbrain dopaminergic neurons, with characteristic neuronal morphology, expressing tyrosine hydroxylase (TH) and microtubule-associated protein 2 (MAP2). There was no evidence of neurodegeneration in the patient lines. A number of disease-specific features were identified, including significantly marked reduction of AADC enzyme activity and dysregulation of the dopaminergic system in patient mDA neurons when compared to the age-matched control. Preliminary data also shows successful lentiviral rescue of the patient-derived mDA cell model. Conclusion: The iPSC-derived mDA neuronal model represents an ideal platform to further elucidate disease mechanisms, as well as to screen novel pharmacological agents for AADC deficiency.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Functional characterisation of aromatic L-amino acid decarboxylase (AADC) deficiency using a patient-derived dopaminergic cell model
Event: UCL (University College London)
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2019. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) Licence (https://creativecommons.org/licenses/by-nc-nd/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL
URI: https://discovery.ucl.ac.uk/id/eprint/10083710
Downloads since deposit
44Downloads
Download activity - last month
Download activity - last 12 months
Downloads by country - last 12 months

Archive Staff Only

View Item View Item