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Dopa-responsive dystonia (DRD) and Parkinson’s disease (PD) are movement disorders caused by the dysfunction of nigrostriatal dopaminergic neurons. Identifying druggable pathways and biomarkers for guiding therapies is crucial due to the debilitating nature of these disorders. Recent genetic studies have identified variants of GTP cyclohydrolase-1 (GCH1), the rate-limiting enzyme in tetrahydrobiopterin (BH4) synthesis, as causative for these movement disorders. Here, we show that genetic and pharmacological inhibition of BH4 synthesis in mice and human midbrain-like organoids accurately recapitulates motor, behavioral and biochemical characteristics of these human diseases, with severity of the phenotype correlating with extent of BH4 deficiency. We also show that BH4 deficiency increases sensitivities to several PD-related stressors in mice and PD human cells, resulting in worse behavioral and physiological outcomes. Conversely, genetic and pharmacological augmentation of BH4 protects mice from genetically- and chemically induced PD-related stressors. Importantly, increasing BH4 levels also protects primary cells from PD-affected individuals and human midbrain-like organoids (hMLOs) from these stressors. Mechanistically, BH4 not only serves as an essential cofactor for dopamine synthesis, but also independently regulates tyrosine hydroxylase levels, protects against ferroptosis, scavenges mitochondrial ROS, maintains neuronal excitability and promotes mitochondrial ATP production, thereby enhancing mitochondrial fitness and cellular respiration in multiple preclinical PD animal models, human dopaminergic midbrain-like organoids and primary cells from PD-affected individuals. Our findings pinpoint the BH4 pathway as a key metabolic program at the intersection of multiple protective mechanisms for the health and function of midbrain dopaminergic neurons, identifying it as a potential therapeutic target for PD.

This thesis describes the application of one and two dimensional proton NMR spectroscopy and associated computational methods to the study of structure in solution of two metalloproteins: the cytochrome subunit (cytPCMH) of Pseudomonas putida para-cresol methylhydroxylase (PCMH) and parsley plastocyanin. Initial work on cytPCMH focused on the haem prosthetic group. Haem meso proton resonances were assigned by combined analysis of NOE data and the PCMH crystal structure. Interproton distances derived from these assignments indicated that one of the axial ligands to the haem Fe, Met 50, has a different orientation in cytPCMH in solution relative to that in crystalline PCMH. Further evidence for this difference between the unbound and bound states of the cytochrome subunit was provided by differences in reduction potentials, and by differences in the pattern of haem methyl group resonances of the oxidised species. Precedents for different ligand orientations among c-type cytochromes have been discussed. Aromatic and methyl group spin system assignment in cytPCMH was carried out as the initial stage of sequential resonance assignment and structure determination. Aromatic side chain assignments, combined with analysis of NOE data, provided further evidence for a local conformational change in cytPCMH relative to PCMH. A potentially important residue in the conformational change was identified. Essentially complete sequential resonance assignments have been obtained for parsley plastocyanin, which has important differences in sequence from other higher plant plastocyanins, and is closely related to some algal plastocyanins. Assignments were initially used in location and characterisation of elements of regular secondary structure. Distance and dihedral angle restraints were then accumulated and used in the calculation of a high resolution three dimensional structure of parsley plastocyanin. This structure has been analysed in detail and compared to reported structures of other plastocyanins. Features of interest include a β-bulge, a cis-Pro bend and a carboxyl---carboxylate hydrogen bond which accounts for parsley plastocyanin's anomalously high pKa . Residue deletions cause elimination of a turn found in most higher plant plastocyanins. This turn is located in an acidic patch binding site, which may be further disrupted in parsley plastocyanin by non-conservative substitution of two charged residues. Calculations show that these sequence differences result in diminution of the negative electrostatic field of parsley plastocyanin relative to that of poplar plastocyanin.