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Midbrain single-cell sequencing to understand Parkinson’s Disease

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Published on Thursday, 17 March 2022

Researchers from the Luxembourg Centre for Systems Biomedicine (LCSB) at the University of Luxembourg, in collaboration with the Max Planck Institute for Molecular Genetics in Berlin, investigated the different cell types present in the human midbrain to better understand their respective role in Parkinson’s disease.  They examined post-mortem samples from people affected by the disease and from healthy individuals, and generated a unique single-nuclei RNA sequencing dataset. Their results, recently published in the scientific journal Brain, revealed the existence of a specific cluster of cells in the midbrain of Parkinson’s patients. They also highlighted the role of glial cells – the non-neuronal cells in the brain – in the pathology of this movement disorder.

Parkinson’s disease in a nutshell

Parkinson’s disease (PD) is characterised by the loss of dopaminergic neurons in the midbrain but the exact causes of the disease are still not well understood. These neurons control brain functions like voluntary movement and behavioural processes such as stress. Their degeneration leads to the disease with its classical symptoms: trembling, stiffness and loss of balance and coordination. However, this is not the whole picture: recent findings indicate that other cell types can also be involved in the course of the disease.

Single-nuclei RNA sequencing of the midbrain

To get information about the role of other non-neuronal cell types, the researchers examined the entire cell population of the midbrain. Sections of post-mortem brain tissues donated by five patients with idiopathic PD, meaning the disease has no known cause in their case, and six healthy controls were examined. The scientists took advantage of the recent developments in single-cell technologies and used an approach called single-nuclei RNA sequencing. “We sequenced 41,000 nuclei in total to obtain a global view of the cell type composition and its contribution to the disease.” explains Prof. Anne Grünewald, head of the Molecular and Functional Neurobiology group at the LCSB and joint-senior author of the article, along with Prof. Malte Spielmann from the Max Planck Institute for Molecular Genetics. This is the first single-nuclei RNA sequencing dataset ever obtained from the idiopathic Parkinson’s disease midbrain.

Discovery of a disease-specific state of neurons

Based on the RNA sequencing results, midbrain cells belonging to the main groups found in the brain - glial, neuronal, ependymal and vascular - were sorted into 12 smaller clusters, indicating 12 different cell types (see illustration below). Each cell type was classified based on the expression of specific marker genes and presents different characteristics. One of the identified clusters corresponds to dopaminergic neurons. These cells are characterised by a specific marker called tyrosine hydroxylase (TH) and were underrepresented in the midbrain tissue of Parkinson’s disease patients.

Left: Cell clusters of neuronal (DaN, excitatory, inhibitory, GABA and CADPS2high), glial (oligodendrocytes, OPCs, microglia, and astrocytes), ependymal and vascular cells (endothelial cells and pericytes).
Right: The proportion of CADPS2high and microglia is much higher (red colour) in IPD.

Interestingly, the researchers also discovered a small population of neuronal cells present in the samples from Parkinson’s patients. This cluster of 120 cells could not be readily classified. “Those cells were almost exclusively found in individuals with Parkinson’s,” points out Semra Smajić, doctoral researcher in the Molecular and Functional Neurobiology group and joint-first author of the article along with Cesar A. Prada-Medina from the Max Planck Institute. “Their sequencing profile is similar to dopaminergic neurons, in the sense that they are the only two cell types that express the CADPS2 gene, but, unlike dopaminergic neurons, they also show a low expression of TH. Taking all that into consideration, we think that this disease-specific cell type might correspond to degenerating dopaminergic neurons.” Further investigations are now needed to confirm this hypothesis.

Reactive microglia: a signature of idiopathic Parkinson’s disease?

As a potential factor in the degeneration of dopaminergic neurons is inflammation, the researchers also took a closer look at inflammatory processes in the brain tissues of Parkinson’s patients and healthy individuals. Their analyses revealed differences in microglial cells, the “cleaning personnel” of the brain that digest pathogens or damaged neurons as part of the overall brain maintenance. They are in charge of the brain’s immune system as well and play a key role in inflammation. 

While resting microglia are ramified, they retract their branches and switch to an amoeboid form when assuming their cleaning role or to a reactive state when performing their function in immune defence. The researchers observed changes in the shape of microglia: the cells were less ramified and more amoeboid in the tissues affected by the disease, indicating their activation. Moreover, the microglia population was overrepresented in the patients’ samples, denoting a higher level of inflammation. Additionally, when trying to classify microglia, the team discovered three sub-populations defined by a high expression of genes involved in the inflammatory response linked to neurodegeneration. “Taken together, our results suggest that microglia contribute to the degeneration of dopaminergic neurons and that glial activation is a central mechanism in idiopathic Parkinson’s disease. It reinforces the relevance of neuroinflammation in the pathology,” explains Prof. Grünewald.

“Past studies have mostly focused on neuronal cells in the midbrain. Our unique single-nuclei RNA sequencing dataset now provides the basis for new research approaches investigating the role of different cell types in the disease and for translational programs that aim to develop immunomodulatory therapies,” concludes Prof. Malte Spielmann.

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