Characterising extracellular ⍺-synuclein and its associated release mechanisms in hiPSC-derived dopamine neurons

Thesis

Parkinson’s disease is neuropathologically characterised by the accumulation of aggregated ⍺-synuclein, a small synaptic protein which is capable of prion-like propagation in its misfolded state. The extracellular release of ⍺-synuclein enables inter-neuronal spread of toxic ⍺-synuclein conformers which precipitates neuronal dysfunction, degeneration and Parkinsonian symptoms. In this work I employ induced pluripotent stem cell-derived dopaminergic neurons to investigate ⍺-synuclein secretion in vitro.

By integrating techniques from cell biology, classical biochemistry and biophysics I have investigated the variability in ⍺-synuclein primary sequence, post-translational modifications and aggregation state in mature neurons from PD patients with SNCA mutations and healthy controls. I show that most ⍺-synuclein transcripts encode the full length 140-amino acid protein however this is truncated to remove the N-terminus in a maturation-dependent manner. By studying stem-cell derived ⍺-synuclein in its native conformation I provide evidence for the presence of high molecular weight ⍺-synuclein conformers in dopaminergic neurons as well as heat labile products of C-terminal truncation of the primary sequence.

Funded by ARUK, I optimised an assay for the amplification of prion-like ⍺-synuclein seeds from conditioned media. This demonstrate that SNCA-triplication and SNCA-A53T mutations increase the release of seeding-competent ⍺-synuclein conformers, a population which may include secreted truncation products. In collaboration with other research groups in Oxford and Cambridge I pilot various single-molecule approaches to study these secreted ⍺-synuclein seeds.

Using CRISPR interference knockdown technology and pharmacological modulators I then interrogate the release mechanisms for ⍺-synuclein in dopaminergic neurons. Through identification of four possible genetic regulators of secretion, I provide the first evidence that SNARE-mediated fusion of autophagic vesicles is essential to ⍺-synuclein secretion from dopaminergic neurons.

Together the data presented in this thesis highlight the value of iPSC-derived neuronal models for the study of ⍺-synuclein biology and pathobiology in the quest for a ⍺-synuclein-directed disease-modifying therapeutic.

Authors: Thomas-Wright, I

• DOI: 10.5287/ora-xvnqepx9n
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Parkinson's disease; protein aggregation; ⍺-synuclein; hiPSC-derived neurons
• Subjects: Molecular Neuroscience; Biochemisty; Stem cell biology