Budinger, Dimitri; (2023) Elucidating the role of brain manganese in health and disease using a patient-derived neuronal model. Doctoral thesis (Ph.D), UCL (University College London).

Text Dimitri Budinger_PhD Thesis_March 2023.pdf - Accepted Version Access restricted to UCL open access staff until 1 May 2025. Download (67MB)

Abstract

Introduction: Manganese (Mn) is an essential trace metal, crucial for normal neuronal cell function. Acquired and genetic disorders leading to Mn imbalance in humans can result in a broad spectrum of phenotypes, including parkinsonism-dystonia and neurodevelopmental delay/regression. Bi-allelic mutations in genes encoding Mn transporters ZIP14, ZIP8 and ZnT10 have been identified in patients with Mn dyshomeostasis, and are associated with progressive, often debilitating disorders. Little is known about the molecular mechanisms underlying Mn dysregulation and neurotoxicity, and there are currently no effective treatments. Aim: The aim of this thesis is to identify disease-specific phenotypes and decipher the molecular mechanisms associated with Mn dyshomeostasis in a patient-derived midbrain dopaminergic (mDA) neuronal model. Methods: Using age-matched and CRISPR-Cas9 generated controls and patient-derived induced pluripotent stem cells, I have developed a humanized mDA neuronal model of the three inherited Mn transportopathies. I have used a range of molecular, cellular, and transcriptomic approaches to investigate the physiological role and pathophysiological sequelae of Mn in health and disease. Results: Key results show disease-specific dysregulation of Mn and other metals. Mn dysregulation also leads to an increase in cellular stress through activation of the SAPK/JNK pathway, which leads to caspase-3 activation and is suggestive of increased apoptosis. Assessment of mitochondrial function highlights dysregulation of mitochondrial OXPHOS complexes, with abnormal gene and protein expression as well as impaired enzymatic activity, defects in mitochondrial membrane potential, and ATP production. Broader bulk RNA-sequencing analysis confirms the observed disease-specific mitochondrial dysregulation as well as dysregulation of pathways associated with collagen metabolism, synaptic function, oxidative stress, and cell death. Bulk-RNA sequencing findings are further confirmed by calcium imaging, Seahorse assay, and an endocytic uptake assay. Moreover, transcriptomic analysis highlights common dysregulated pathways between these Mn transportopathies and other similar neurodegenerative disorders such as Parkinson’s and Alzheimer’s disease.

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