Midbrain organoids with anSNCAgene triplication model key features of synucleinopathy

Abstract

SNCA, the first gene associated with Parkinson’s disease, encodes the α-synuclein protein, the predominant component within pathological inclusions termed Lewy bodies. The presence of Lewy bodies is one of the classical hallmarks found in the brain of patients with Parkinson’s disease, and Lewy bodies have also been observed in patients with other synucleinopathies. However, the study of α-synuclein pathology in cells has relied largely on two-dimensional culture models, which typically lack the cellular diversity and complex spatial environment found in the brain. Here, to address this gap, we use 3D midbrain organoids, differentiated from human induced pluripotent stem cells derived from patients carrying a triplication of the SNCA gene and from CRISPR/Cas9 corrected isogenic control iPSCs. These human midbrain organoids recapitulate key features of α-synuclein pathology observed in the brains of patients with synucleinopathies. In particular, we find that SNCA triplication human midbrain organoids express elevated levels of α-synuclein and exhibit an age-dependent increase in α-synuclein aggregation, manifested by the presence of both oligomeric and phosphorylated forms of α-synuclein. These phosphorylated α-synuclein aggregates were found in both neurons and glial cells and their time-dependent accumulation correlated with a selective reduction in dopaminergic neuron numbers. Thus, human midbrain organoids from patients carrying SNCA gene multiplication can reliably model key pathological features of Parkinson’s disease and provide a powerful system to study the pathogenesis of synucleinopathies.

An Approach to Measuring Protein Turnover in Human Induced Pluripotent Stem Cell Organoids by Mass Spectrometry

“…This technology serves as a critical bridge between 2D cultures and in vivo models in examining complex neural mechanisms and their dysregulation in disease. Unlike traditional in vitro cultures, the architecture of organoids consists of multiple region-specific cell types conferring more physiologically relevant characteristics [5,6]. Two examples of disease-relevant hallmarks in organoids that remain poorly captured in 2D culture models are as follows:…”

“…(1) midbrain organoids are capable of producing neuromelanin-like granules, a distinct structure resulting from dopamine synthesis that are highly enriched in the neurons lost in Parkinson's disease (PD) [1,5,7], and (2) β-amyloid plaques and neurofibrillary tangles are found in cerebral organoids, a pathological marker of Alzheimer's disease (AD) pathology [8].…”

“…Other studies have validated the use of heavy leucine in turnover measurements by feeding Drosophila melanogaster [5,5,5]-deuterium-3-leucine (D3-Leu) food that was incorporated into the flies over time [24]. Likewise, organoid models can be cultured with 13 C, 15 N-labeled lysine and arginine to characterize growth and protein abundance under different conditions [25].…”

“…This technology serves as a critical bridge between 2D cultures and in vivo models in examining complex neural mechanisms and their dysregulation in disease. Unlike traditional in vitro cultures, the architecture of organoids consists of multiple region-specific cell types conferring more physiologically relevant characteristics [5,6]. Two examples of disease-relevant hallmarks in organoids that remain poorly captured in 2D culture models are as follows: (1) midbrain organoids are capable of producing neuromelanin-like granules, a distinct structure resulting from dopamine synthesis that are highly enriched in the neurons lost in Parkinson’s disease (PD) [1,5,7], and (2) β-amyloid plaques and neurofibrillary tangles are found in cerebral organoids, a pathological marker of Alzheimer’s disease (AD) pathology [8].…”

“…Select the row and set the identifier rule to "Uniprot identifier". The minimum peptide length can be left at 7 a.a. 5. In "Identification", ensure that you select "Match between runs" to enable transferring of protein identifications across runs.…”