Single-cell sequencing of human midbrain reveals glial activation and a Parkinson-specific neuronal state.

in Brain : a journal of neurology (2022), 145(3), 964-978

Idiopathic Parkinson's disease is characterized by a progressive loss of dopaminergic neurons, but the exact disease etiology remains largely unknown. To date, Parkinson's disease research has mainly ... [more ▼]

Idiopathic Parkinson's disease is characterized by a progressive loss of dopaminergic neurons, but the exact disease etiology remains largely unknown. To date, Parkinson's disease research has mainly focused on nigral dopaminergic neurons, although recent studies suggest disease-related changes also in non-neuronal cells and in midbrain regions beyond the substantia nigra. While there is some evidence for glial involvement in Parkinson's disease, the molecular mechanisms remain poorly understood. The aim of this study was to characterize the contribution of all cell types of the midbrain to Parkinson's disease pathology by single-nuclei RNA sequencing and to assess the cell type-specific risk for Parkinson's disease employing the latest genome-wide association study. We profiled >41 000 single-nuclei transcriptomes of postmortem midbrain from six idiopathic Parkinson's disease patients and five age-/sex-matched controls. To validate our findings in a spatial context, we utilized immunolabeling of the same tissues. Moreover, we analyzed Parkinson's disease-associated risk enrichment in genes with cell type-specific expression patterns. We discovered a neuronal cell cluster characterized by CADPS2 overexpression and low TH levels, which was exclusively present in IPD midbrains. Validation analyses in laser-microdissected neurons suggest that this cluster represents dysfunctional dopaminergic neurons. With regard to glial cells, we observed an increase in nigral microglia in Parkinson's disease patients. Moreover, nigral idiopathic Parkinson's disease microglia were more amoeboid, indicating an activated state. We also discovered a reduction in idiopathic Parkinson's disease oligodendrocyte numbers with the remaining cells being characterized by a stress-induced upregulation of S100B. Parkinson's disease risk variants were associated with glia- and neuron-specific gene expression patterns in idiopathic Parkinson's disease cases. Furthermore, astrocytes and microglia presented idiopathic Parkinson's disease-specific cell proliferation and dysregulation of genes related to unfolded protein response and cytokine signaling. While reactive patient astrocytes showed CD44 overexpression, idiopathic Parkinson's disease-microglia revealed a pro-inflammatory trajectory characterized by elevated levels of IL1B, GPNMB, and HSP90AA1. Taken together, we generated the first single-nuclei RNA sequencing dataset from the idiopathic Parkinson's disease midbrain, which highlights a disease-specific neuronal cell cluster as well as 'pan-glial' activation as a central mechanism in the pathology of the movement disorder. This finding warrants further research into inflammatory signaling and immunomodulatory treatments in Parkinson's disease. [less ▲]

Nanopore Single-Molecule Sequencing for Mitochondrial DNA Methylation Analysis: Investigating Parkin-Associated Parkinsonism as a Proof of Concept

in Frontiers in Aging Neuroscience (2021)

Objective: To establish a workflow for mitochondrial DNA (mtDNA) CpG methylation using Nanopore whole-genome sequencing and perform first pilot experiments on affected Parkin biallelic mutation carriers ... [more ▼]

Objective: To establish a workflow for mitochondrial DNA (mtDNA) CpG methylation using Nanopore whole-genome sequencing and perform first pilot experiments on affected Parkin biallelic mutation carriers (Parkin-PD) and healthy controls. Background: Mitochondria, including mtDNA, are established key players in Parkinson's disease (PD) pathogenesis. Mutations in Parkin, essential for degradation of damaged mitochondria, cause early-onset PD. However, mtDNA methylation and its implication in PD is understudied. Herein, we establish a workflow using Nanopore sequencing to directly detect mtDNA CpG methylation and compare mtDNA methylation between Parkin-related PD and healthy individuals. Methods: To obtain mtDNA, whole-genome Nanopore sequencing was performed on blood-derived from five Parkin-PD and three control subjects. In addition, induced pluripotent stem cell (iPSC)-derived midbrain neurons from four of these patients with PD and the three control subjects were investigated. The workflow was validated, using methylated and unmethylated 897 bp synthetic DNA samples at different dilution ratios (0, 50, 100% methylation) and mtDNA without methylation. MtDNA CpG methylation frequency (MF) was detected using Nanopolish and Megalodon. Results: Across all blood-derived samples, we obtained a mean coverage of 250.3X (SD ± 80.5X) and across all neuron-derived samples 830X (SD ± 465X) of the mitochondrial genome. We detected overall low-level CpG methylation from the blood-derived DNA (mean MF ± SD = 0.029 ± 0.041) and neuron-derived DNA (mean MF ± SD = 0.019 ± 0.035). Validation of the workflow, using synthetic DNA samples showed that highly methylated DNA molecules were prone to lower Guppy Phred quality scores and thereby more likely to fail Guppy base-calling. CpG methylation in blood- and neuron-derived DNA was significantly lower in Parkin-PD compared to controls (Mann-Whitney U-test p < 0.05). Conclusion: Nanopore sequencing is a useful method to investigate mtDNA methylation architecture, including Guppy-failed reads is of importance when investigating highly methylated sites. We present a mtDNA methylation workflow and suggest methylation variability across different tissues and between Parkin-PD patients and controls as an initial model to investigate. [less ▲]

Astrocyte-Neuron Metabolic Crosstalk in Neurodegeneration: A Mitochondrial Perspective.

in Frontiers in endocrinology (2021), 12

Converging evidence made clear that declining brain energetics contribute to aging and are implicated in the initiation and progression of neurodegenerative disorders such as Alzheimer's and Parkinson's ... [more ▼]

Converging evidence made clear that declining brain energetics contribute to aging and are implicated in the initiation and progression of neurodegenerative disorders such as Alzheimer's and Parkinson's disease. Indeed, both pathologies involve instances of hypometabolism of glucose and oxygen in the brain causing mitochondrial dysfunction, energetic failure and oxidative stress. Importantly, recent evidence suggests that astrocytes, which play a key role in supporting neuronal function and metabolism, might contribute to the development of neurodegenerative diseases. Therefore, exploring how the neuro-supportive role of astrocytes may be impaired in the context of these disorders has great therapeutic potential. In the following, we will discuss some of the so far identified features underlining the astrocyte-neuron metabolic crosstalk. Thereby, special focus will be given to the role of mitochondria. Furthermore, we will report on recent advancements concerning iPSC-derived models used to unravel the metabolic contribution of astrocytes to neuronal demise. Finally, we discuss how mitochondrial dysfunction in astrocytes could contribute to inflammatory signaling in neurodegenerative diseases. [less ▲]