Midbrain organoids with anSNCAgene triplication model key features of synucleinopathy

Abstract

SNCA, the first gene associated with Parkinson′s disease, encodes the α-synuclein (α-syn) protein, the predominant component within pathological inclusions termed Lewy bodies (LBs). The presence of LBs is one of the classical hallmarks found in the brain of patients with Parkinson′s disease, and LBs have also been observed in patients with other synucleinopathies. However, the study of α-syn 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 (hMOs), 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 hMOs recapitulate key features of α-syn pathology observed in the brains of patients with synucleinopathies. In particular, we find that SNCA triplication hMOs express elevated levels of α-syn and exhibit an age-dependent increase in α-syn aggregation, manifested by the presence of both oligomeric and phosphorylated forms of α-syn. These phosphorylated α-syn aggregates were found in both neurons and glial cells and their time-dependent accumulation correlated with a selective reduction in dopaminergic neuron numbers. Thus, hMOs 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.

Microfabricated disk technology: rapid scale up in midbrain organoid generation

“…For detailed protocols, please refer to our previous publications [7, 47]. Briefly, hMOs were fixed and cryosectioned according to our published methods [7].…”

Fast 3D Optoacoustic Mesoscopy of Neuromelanin Through Entire Human Midbrain Organoids at Single‐Cell Resolution

“…Fontana Masson staining was performed in 2 consecutive sections at 0-200, 200-400, and 400-600 μm deep, using a stain kit (Melanin Stain, #ab150669, Abcam, Cambridge, UK), following the manufacturer's instructions. [8] Full sections were then imaged with the ZEISS Stemi 508 stereomicroscope combined with the ZEISS Axiocam ERc 5 s camera (Zeiss, Jena, DE). Images were acquired with a clinical Olympus BX46 (Olympus, Tokyo, JP) microscope and an Olympus DP27 digital colour camera obtaining Red, Green, and Blue (RGB) images.…”

Alpha synuclein determines ferroptosis sensitivity in dopaminergic neurons via modulation of ether-phospholipid membrane composition

“…Control smNPC line C4 -WT was previously described and characterised in (Boussaad et al, 2020), and C4 -SNCA KO iPSC were generated by transfection with the Cas9 plasmid pX330 (Addgene,42,230). The SNCA triplication line was obtained from EBISC (cell line Edi001-A) and the isogenic gene corrected line (Iso WT) was generated and kindly provided by Dr Tilo Kunath from the University of Edinburgh (Mohamed et al, 2021).…”