Abela-Arnold, Lucia; (2024) DNAJC6-related Parkinsonism: Disease Characterisation and Development of a Patient-Derived Dopaminergic Neuronal Cell Model. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

INTRODUCTION: Biallelic loss-of-function mutations in DNAJC6 cause a complex early-onset neurodegenerative condition characterised by juvenile-onset parkinsonism-dystonia with additional neurological, neurodevelopmental and neuropsychiatric features. The disease course is rapidly progressive and usually leads to loss of ambulation in mid-adolescence. DNAJC6 encodes auxilin, a neuron-specific co-chaperone protein involved in clathrin-mediated endocytosis at presynaptic terminals. To date, the underlying disease mechanisms have not yet been fully elucidated and there are no disease-modifying treatments. AIM: The aim of this PhD project is to (i) provide comprehensive clinical characterisation of a DNAJC6 patient cohort and (ii) develop a patient-derived midbrain dopaminergic (mDA) neuronal cell model to investigate molecular disease mechanisms and develop a proof-of-concept gene therapy approach. METHODS: I generated induced pluripotent stem cells (iPSC) from four patients with pathogenic recessive mutations in DNAJC6 and then differentiated them into mDA neurons along with two age-matched controls and one CRISPR-corrected isogenic control. Using this humanised neuronal cell model, I performed a number of cellular, molecular and transcriptomic assays to further elucidate the role of auxilin deficiency in DNAJC6-related disease. RESULTS: Loss of auxilin expression with impairment of clathrin-mediated endocytosis and disturbance of synaptic vesicle homeostasis are key disease-specific phenotypic findings in the mDA neuronal cell model. Disease-specific defects in ventral midbrain patterning were observed, with reduced LMX1A/FOXA2 co-expression at early stages of dopaminergic differentiation. Terminally differentiated mDA neurons showed decreased staining of the late neuronal marker NeuN and reduced primary neurite outgrowth, suggesting disturbances in neuronal maturation. Transcriptomic analysis using bulk RNA-seq analysis showed disturbance of presynaptic membrane processes and synaptic vesicle cycling as well as dysregulation of numerous neurodevelopmental processes. Transfection of mDA neurons with a lentiviral vector containing the wild-type DNAJC6 gene restored auxilin protein levels and improved clathrin-mediated endocytosis and thus provides initial proof-of-concept for a gene therapy approach for this condition.

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