Protein aggregation and calcium dysregulation are the earliest hallmarks of synucleinopathy in human midbrain dopaminergic neurons

Mutations in the SNCA gene cause autosomal dominant Parkinson’s disease (PD), with progressive loss of dopaminergic neurons in the substantia nigra, and accumulation of aggregates of α-synuclein. However, the sequence of molecular events that proceed from the SNCA mutation during development, to its end stage pathology is unknown. Utilising human induced pluripotent stem cells (hiPSCs) with SNCA mutations, we resolved the temporal sequence of pathophysiological events that occur during neuronal differentiation in order to discover the early, and likely causative, events in synucleinopathies. We adapted a small molecule-based protocol that generates highly enriched midbrain dopaminergic (mDA) neurons (>80%). We characterised their molecular identity using single-cell RNA sequencing and their functional identity through the synthesis and secretion of dopamine, the ability to generate action potentials, and form functional synapses and networks. RNA velocity analyses confirmed the developmental transcriptomic trajectory of midbrain neural precursors into mDA neurons using our approach, and identified key driver genes in mDA neuronal development. To characterise the synucleinopathy, we adopted super-resolution methods to determine the number, size and structure of aggregates in SNCA -mutant mDA neurons. At one week of differentiation, prior to maturation to mDA neurons of molecular and functional identity, we demonstrate the formation of small aggregates; specifically, β-sheet rich oligomeric aggregates, in SNCA -mutant midbrain immature neurons. The aggregation progresses over time to accumulate phosphorylated aggregates, and later fibrillar aggregates. When the midbrain neurons were functional, we observed evidence of impaired physiological calcium signalling, with raised basal calcium, and impairments in cytosolic and mitochondrial calcium efflux. Once midbrain identity fully developed, SNCA -mutant neurons exhibited bioenergetic impairments, mitochondrial dysfunction and oxidative stress. During the maturation of mDA neurons, upregulation of mitophagy and autophagy occured, and ultimately these multiple cellular stresses lead to an increase in cell death by six weeks post-differentiation. Our differentiation paradigm generates an efficient model for studying disease mechanisms in PD, and highlights that protein misfolding to generate intraneuronal oligomers is one of the earliest critical events driving disease in human neurons, rather than a late-stage hallmark of the disease. ### Competing Interest Statement The authors have declared no competing interest.