Protein aggregation and calcium dysregulation are hallmarks of familial Parkinson’s disease in midbrain dopaminergic neurons

Abstract

Mutations in the SNCA gene cause autosomal dominant Parkinson’s disease (PD), with loss of dopaminergic neurons in the substantia nigra, and aggregation of α-synuclein. The sequence of molecular events that proceed from an SNCA mutation during development, to end-stage pathology is unknown. Utilising human-induced pluripotent stem cells (hiPSCs), we resolved the temporal sequence of SNCA-induced pathophysiological events in order to discover early, and likely causative, events. Our small molecule-based protocol generates highly enriched midbrain dopaminergic (mDA) neurons: molecular identity was confirmed using single-cell RNA sequencing and proteomics, and functional identity was established through dopamine synthesis, and measures of electrophysiological activity. At the earliest stage of differentiation, prior to maturation to mDA neurons, we demonstrate the formation of small β-sheet-rich oligomeric aggregates, in SNCA-mutant cultures. Aggregation persists and progresses, ultimately resulting in the accumulation of phosphorylated α-synuclein aggregates. Impaired intracellular calcium signalling, increased basal calcium, and impairments in mitochondrial calcium handling occurred early at day 34–41 post differentiation. Once midbrain identity fully developed, at day 48–62 post differentiation, SNCA-mutant neurons exhibited mitochondrial dysfunction, oxidative stress, lysosomal swelling and increased autophagy. Ultimately these multiple cellular stresses lead to abnormal excitability, altered neuronal activity, and cell death. 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.

Alpha-synuclein in Parkinson’s disease and other synucleinopathies: from overt neurodegeneration back to early synaptic dysfunction

“…Oligomers produced during secondary surface nucleation induce the permeabilization of cell membranes through to their lipophilic properties. Associated with these events, higher basal levels of cytosolic Ca 2+ , increased Ca 2+ influx upon stimulation, and delayed recovery of Ca 2+ levels in neurons are then observed since the early phases, leading to multiple Ca 2+ homeostasis alterations that persist with disease progression [ 18 ].…”

“…of cell membranes through to their lipophilic properties. Associated with these events, higher basal levels of cytosolic Ca 2+ , increased Ca 2+ influx upon stimulation, and delayed recovery of Ca 2+ levels in neurons are then observed since the early phases, leading to multiple Ca 2+ homeostasis alterations that persist with disease progression [18]. Native α-syn is present in synaptic terminals, in the nucleus of neuronal cells [19], mitochondria [20], endoplasmic reticulum (ER) [21], Golgi apparatus (GA) [22], and in the endolysosomal system [23].…”

Diverging Parkinson’s Disease Pathology between patient-derivedGBAN370S, LRRK2G2019Sand engineeredSNCAA53TiPSC-derived Dopaminergic Neurons

“…Conversely, LRRK2 G2019S mutant neurons had faster calcium oscillations which aligned with the hyperexcitable phenotype (increased network burst frequency) observed on MEA activity. One possible mechanism underlying this observation is that high expression of α-Syn has been shown to disrupt calcium homeostasis and potentially alter neuronal activity 50 . This is supported by our observation of increased α-Syn accumulation in all mutant PD disease lines.…”

Developmental pathways linked to the vulnerability of adult midbrain dopaminergic neurons to neurodegeneration

“…Almost nothing is known about the electrophysiological activities and Ca 2+ homeostatic processes in the emerging mDA neurons, most likely playing a role already during the earliest stages of mDA neuron development and long before they have established a proper connectivity with their efferent and afferent targets, although their mitochondrial energy metabolism and autophagic/lysosomal clearance of misfolded proteins and defective organelles are now beginning to be unraveled in the human context in vitro ( section 3.1.2 ). This fact has probably led to a collective neglect of these aspects arguing that they might not be biologically relevant, although several evidences have meanwhile accumulated suggesting the opposite is true for the developing mammalian VM and mDA neurons, both in the wildtype and in the PD context ( Chan et al, 2007 ; Rockhill et al, 2009 ; Ferrari et al, 2012 ; Ramirez-Latorre, 2012 ; Kim et al, 2020 ; Carola et al, 2021 ; Akrioti et al, 2022 ; Stern et al, 2022 ; Virdi et al, 2022 ). It has been suggested that such ion- and neurotransmitter-driven early (immature) activities in developing neurons represent “phenotypic checkpoints” integrating intrinsic TF-mediated (genetic) developmental pathways with extrinsic signals from the surrounding environment to ensure the overall correct establishment of a neuron’s identity and function in the broader context of the nervous system ( Ben-Ari and Spitzer, 2010 ).…”