Visualising the physiological and pathological dynamics of alpha-synuclein protein in human pluripotent stem cell derived cortical neurons

Abstract

Dementia with Lewy bodies and Parkinson’s disease are part of a family of progressive neurodegenerative diseases known as alpha synucleinopathies, of which the protein alpha-synuclein (αSyn) is a major pathological driver. Whilst αSyn’s role in neurodegeneration is extensively studied, there is much less known about αSyn’s functional role in neurons and whether its normal physiological protein dynamics could lead to neurodegeneration. In this thesis, I investigated αSyn’s physiological role in innate immunity, developed live-imaging cellular models to observe αSyn protein dynamics, and explored how it could transition to a neurotoxic state. We first optimised the differentiation of human pluripotent stem cells (hPSC) into cortical neurons using the dual SMAD inhibition protocol. Application of existing 2D-based differentiation protocols frequently produced highly proliferative non-neuronal cells that significantly affected neuronal culture integrity. We found that pro-collagen I alpha I (COL1A1) robustly labelled these disruptive subpopulations and found that low cell density encouraged COL1A1+ cell proliferation. We showed that COL1A1+ cells co-express neuronal progenitor markers indicating they are derived from a neural cell type, but they do not differentiate to desired neuronal cells. Using COL1A1 as a marker and image quantification, we employed whole well immunostaining to optimise the existing dual SMAD inhibition protocols. We discovered multiple optimisation steps that significantly reduced or eliminated the emergence of COL1A1+ cells. Our improved protocol has decreased batch-to-batch variability and increased the robustness of our modelling. Using our optimised protocol, we investigated αSyn’s normal role in cortical neurons. We and others have shown that αSyn’s physiological role may be in protecting neurons from neurotrophic viral infection. To investigate potential mechanisms of action of αSyn’s innate immune role, we characterised αSyn transcription, protein expression, and subcellular localisation under TLR3 agonism and Type-I interferon stimulation. We first showed that cortical neurons exhibited a robust transcriptional response to innate immune stimulation. We observed that acute Type-I interferon stimulation decreased intracellular αSyn protein levels and secretion, but did not change αSyn transcriptional levels in cortical neurons. We found that αSyn-null neurons exhibited delayed transcriptional responses to acute Type-I interferon stimulation, indicating that αSyn protein dynamics may support immediate-early anti-viral responses. We also found that αSyn exhibits a heterogeneous subcellular localisation indicative of a highly dynamic protein, and that Type-I interferon may promote αSyn nuclear localisation in a subset of cortical neurons. To further investigate αSyn’s dynamic role in real time, we constructed N-terminal and C-terminal αSyn-HaloTag live reporter constructs. We stably integrated N-terminal, C-terminal αSyn-HaloTag and HaloTag-only transgenes into the AAVS1 locus in multiple hPSC lines. Using live imaging microscopy and fluorescence-lifetime imaging microscopy (FLIM), we found that N-terminal and C-terminal constructs exhibited similar subcellular localisation profiles to endogenous αSyn in hPSCs but observed significantly increased fluorescence with our C-terminal αSyn-HaloTag construct. We also successfully characterised our C-terminal αSyn-HaloTag construct in mature cortical neurons and demonstrated that endogenously expressed αSyn-HaloTag fusion proteins could interact with exogenous αSyn-647 oligomers during live imaging. Whilst we could not elucidate a clear mechanism of action regarding αSyn’s role in innate immunity, we successfully developed a set of live imaging αSyn constructs and an optimised differentiation protocol to study αSyn protein dynamics in cortical neurons. This novel research provides a powerful platform to investigate αSyn physiological and pathological protein dynamics at high fidelity and has powerful future applications for synucleiopathy disease modelling research and drug screening