Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the accumulation and aggregation of the synaptic protein alpha-synuclein (aSyn), and the death of dopaminergic neurons in the substantia nigra pars compacta. These neurons primarily project to the striatum, forming the nigrostriatal pathway crucial for voluntary movement control. Despite extensive research, the precise pathophysiological mechanisms underlying neurodegeneration in PD remain elusive, partly due to the absence of a disease model capable of accurately replicating biological processes at the cellular and tissue levels. Existing experimental models for studying PD include in vivo animal models involving various organisms, such as flies, rodents, and non-human primates. While these models are valuable, they fail to fully mimic the complex genetic background observed in human cells and do not spontaneously develop a PD-like phenotype without external manipulation. Therefore, there is a critical need to explore PD using human dopaminergic neurons to gain deeper insights into the disease mechanisms.

The discovery that somatic cells can be reprogrammed has revolutionized biomedical research. This breakthrough enables us to take cells from individuals, turn them into stem cells, and then transform them into neurons. These reprogrammed cells, called induced pluripotent stem cells (iPSCs), provide a valuable tool for developing stronger human-based in vitro models without the ethical concerns linked to embryonic stem cells. However, many of the existing in vitro models have been established in two-dimensional or monolayer cultures, which fail to capture the intricate interactions between neurons and other brain cells, such as glia. To address this limitation, researchers have turned to three-dimensional structures called organoids. These miniature cell masses can replicate key features of specific brain regions or human tissues, providing a more physiologically relevant environment for studying disease mechanisms. More recently, a ground-breaking advancement known as assembloids has emerged. Assembloids are created by fusing two or more region-specific organoids, enabling the creation of complex neuronal connections between different brain regions within a single model system. This innovative approach holds great promise for advancing our understanding of neurological disorders, including PD, by faithfully recapitulating the intricate cellular interactions found in the human brain.

Our project aims to create an in vitro model of PD using a midbrain-striatal assembloid. This model seeks to replicate both the circuitry and connections of the nigrostriatal pathway, as well as key features of synucleinopathy, a hallmark of PD. To achieve this, we developed midbrain dopaminergic (MO) and striatal GABAergic (SO) organoids from human iPSCs carrying PD-related mutations or their isogenic controls. First, we confirmed the identity of MO and SO organoids by staining for specific markers of midbrain dopaminergic and striatal GABAergic neurons using immunofluorescence. Then, to track the development of projections within the assembloid, we labeled each organoid with a fluorescent marker before fusing them. Our analysis showed the presence of projections from the dopaminergic midbrain side of the assembloid to the GABAergic striatal side as early as 15 days after fusion, with an increase in projections observed over time. Furthermore, we modeled synucleinopathy by introducing preformed fibrils of aSyn into the assembloid. These fibrils act as seeds for the aggregation of endogenous aSyn. Our results demonstrated a temporal increase in aggregated aSyn, along with a decrease in dopaminergic neurons in the midbrain side of the assembloid, mimicking key pathophysiological characteristics of PD. In summary, we have successfully developed a midbrain-striatal assembloid capable of replicating regional features of the nigrostriatal pathway and the main pathological hallmarks of PD. This innovative human cell model holds great potential for advancing our understanding of PD pathogenesis, exploring potential therapeutic strategies, and testing neuroprotective treatments in a human-relevant system.

Beatriz Elena Lucumí Villegas, MD she is a PhD student at CERVO, Université Laval, Quebec City, Quebec, Canada. She is part of the Martin Lévesque's lab. She was a poster presenter at the WPC 2023.

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