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 ▲]

PINK1 deficiency impairs adult neurogenesis of dopaminergic neurons

in Scientific Reports (2021)

Mitochondrial and Clearance Impairment in p.D620N VPS35 Patient-Derived Neurons

in Movement Disorders (2020)

Background: VPS35 is part of the retromer complex and is responsible for the trafficking and recycling of proteins implicated in autophagy and lysosomal degradation, but also takes part in the degradation ... [more ▼]

Background: VPS35 is part of the retromer complex and is responsible for the trafficking and recycling of proteins implicated in autophagy and lysosomal degradation, but also takes part in the degradation of mitochondrial proteins via mitochondria-derived vesicles. The p.D620N mutation of VPS35 causes an autosomal-dominant form of Parkinson’s disease (PD), clinically representing typical PD. Objective: Most of the studies on p.D620N VPS35 were performed on human tumor cell lines, rodent models overexpressing mutant VPS35, or in patient-derived fibroblasts. Here, based on identified target proteins, we investigated the implication of mutant VPS35 in autophagy, lysosomal degradation, and mitochondrial function in induced pluripotent stem cell-derived neurons from a patient harboring the p.D620N mutation. Methods: We reprogrammed fibroblasts from a PD patient carrying the p.D620N mutation in the VPS35 gene and from two healthy donors in induced pluripotent stem cells. These were subsequently differentiated into neuronal precursor cells to finally generate midbrain dopaminergic neurons. Results: We observed a decreased autophagic flux and lysosomal mass associated with an accumulation of α-synuclein in patient-derived neurons compared to controls. Moreover, patient-derived neurons presented a mitochondrial dysfunction with decreased membrane potential, impaired mitochondrial respiration, and increased production of reactive oxygen species associated with a defect in mitochondrial quality control via mitophagy. Conclusion: We describe for the first time the impact of the p.D620N VPS35 mutation on autophago-lysosome pathway and mitochondrial function in stem cell-derived neurons from an affected p.D620N carrier and define neuronal phenotypes for future pharmacological interventions [less ▲]

Synapse alterations precede neuronal damage and storage pathology in a human cerebral organoid model of CLN3-juvenile neuronal ceroid lipofuscinosis

in Acta Neuropathologica Communications (2020)

Impaired Mitochondrial-Endoplasmic Reticulum Interaction and Mitophagy in Miro1-Mutant Neurons in Parkinson’s Disease

in Human Molecular Genetics (2020)

Deep Learning Quality Control for High-Throughput High-Content Screening Microscopy Images

Poster (2019, October 10)

Automation of biological image analysis is essential to boost biomedical research. The study of complex diseases such as neurodegenerative diseases calls for big amounts of data to build models towards ... [more ▼]

Automation of biological image analysis is essential to boost biomedical research. The study of complex diseases such as neurodegenerative diseases calls for big amounts of data to build models towards precision medicine. Such data acquisition is feasible in the context of high-throughput high-content screening (HTHCS) in which the quality of the results relays on the accuracy of image analysis. Deep learning (DL) yields great performance in image analysis tasks especially with big amounts of data such as the produced in HTHCS contexts. Such DL and HTHCS strength is also their biggest weakness since DL solutions are highly sensitive to bad quality datasets. Hence, accurate Quality Control (QC) for microscopy HTHCS becomes an essential step to obtain reliable pipelines for HTHCS analysis. Usually, artifacts found on these platforms are the consequence of out-of-focus and undesirable density variations. The importance of accurate outlier detection becomes essential for both the training process of generic ML solutions (i.e. segmentation or classification) and the QC of the input data such solution will predict on. Moreover, during the QC of the input dataset, we aim not only to discard unsuitable images but to report the user on the quality of its dataset giving the user the choice to keep or discard the bad images. To build the QC solution we employed fluorescent microscopy images of rosella biosensor generated in the HTHCS platform. A total of 15 planes ranging from -6z to +7z steps to the two optimum planes. We evaluated 27 known focus measure operators and concluded that they have low sensitivity in noisy conditions. We propose a CNN solution which predicts the focus error based on the distance to the optimal plane, outperforming the evaluated focus operators. This QC allows for better results in cell segmentation models based on U-Net architecture as well as promising improvements in image classification tasks. [less ▲]

Guidelines for Fluorescent Guided Biallelic HDR Targeting Selection With PiggyBac System Removal for Gene Editing

in Frontiers in Genetics (2019)

Impaired serine metabolism complements LRRK2-G2019S pathogenicity in PD patients

in Parkinsonism and Related Disorders (2019)

Automated microfluidic cell culture of stem cell derived dopaminergic neurons

in Scientific Reports (2019)

THE PARKINSON’S DISEASE ASSOCIATED PINK1-PARKIN PATHWAY IN PATHOLOGY AND DEVELOPMENT

Doctoral thesis (2018)

Parkinson’s disease (PD) has an aetiology not completely understood. One of the hypothesis in the field is that many neurodegenerative diseases are influenced by developmental disorders. The underlying ... [more ▼]

Parkinson’s disease (PD) has an aetiology not completely understood. One of the hypothesis in the field is that many neurodegenerative diseases are influenced by developmental disorders. The underlying concept is that already during brain development some processes are deregulated producing a higher degree of susceptibility for neurodegeneration during aging. Two hereditary early onset forms of PD are caused by recessive mutations in PTEN-induced putative kinase 1 (PINK1) and Parkin genes that regulate mitochondrial function and morphology, quarantining damaged mitochondria before their degradation as well as triggering the process of mitophagy. Our hypothesis is that alterations of the Pink1-Parkin pathway have an impact in mitochondrial physiology tempering the differentiation ability of neuroepithelial stem cells into dopaminergic neurons. For evaluating this hypothesis we reprogramed patients’ fibroblasts carrying PINK1 mutations, as well as from healthy individuals, to human induced pluripotent stem cells. We developed a streamlined technique of gene editing (FACE) by using the CRISPR/Cas9 system combined with a composite of fluorescent proteins in the donor template for biallelic gene targeting. Isogenic controls were generated using this technique that allowed us to analyze the contribution of corrected patients’ mutations in the cellular defects observed. Human iPSCs were differentiated into a neuroepithelial stem cell state (NESC) from where the cells were further differentiated into neurons. We established different algorithms for pattern recognition and applied them for image analysis of different features such as mitochondrial morphology, proliferation capacity, apoptosis and differentiation. Patient’s derived cells presented an impaired differentiation efficiency into dopaminergic neurons as well as an imbalanced cell renewal that can be linked to the mitochondrial differences. Using 3D cultures, such as microfluidics and organoids, we were able to recapitulate this differentiation impairment in a system that mimics better the context of an in vivo environment. We evaluated the energetic capabilities of the NESCs and the firing activity of differentiated neurons, which also showed a dysregulation in patient cells. We introduced a new system for large-scale analysis of the autophagy and mitophagy pathways by the combination of stably integrated Rosella constructs in different patients’ lines and an image analysis script for classification of the different subcellular structures involved in these pathways activities. This revealed that the basal activity as well as the response against stressors of these pathways are altered in cells derived from patients having different mutations causative of PD. We performed a screen of repurposed drugs as well as of novel compounds to evaluate their impact in this altered developmental transition identifying a potential candidate to be further analysed in an in vivo context. [less ▲]