Research projects

This section introduces all current and prospective projects of the Molecular & Functional Neurobiology group.

Mitochondrial-based stratification of idiopathic Parkinson’s disease (IPD) patients

Idiopathic Parkinson’s disease (IPD) is a complex condition, in which a genetic cause has not been defined, and thus encompasses patients with diverse underlying disease mechanisms. Mitochondrial dysfunction was implicated in PD leading to impaired energy metabolism in dopaminergic neurons of the substantia nigra (SN). In this project, we are assessing the value of mitochondrial-based stratification approaches to help disentangle the complexity of PD. Our study aims at identifying approaches to categorize IPD patients into homogeneous subgroups that will facilitate mechanistic studies, and drug screening ventures.

In collaboration with the Biomedical Data Science group, we have explored published genetic and functional datasets from IPD samples of the FOUNDIN-PD study. RNA-seq data of iPSC-derived 65d-old dopaminergic neurons show that the abundance of a gene associated to familial PD can be used to cluster IPD patients into two subgroups, which present distinctive mitochondrial pathways signatures. Identified altered pathways are being explored in an independent cohort comprising iPSCs from seven IPD cases and four age-matched controls. We are investigating parameters such as mtDNA maintenance, mitochondrial morphology and mitophagy in 2D neuronal cultures. Cluster and machine learning analyses of these results identified a subclass of three IPD patients depicting aberrant mitochondrial function. Together with the Developmental & Cellular Biology group, we are exploring the implication of our findings in more complex models such as midbrain organoids, generated from the same cohort. We are evaluating cell type dynamics and viability, neuronal development and function as well as astrocyte function. Based on our findings, we are investigating possible links between mitochondrial dysfunction and astroglial activation. This project (“Model-IPD”) is funded through the ATTRACT program of the Luxembourg National Research Fund (FNR).

Single-cell transcriptomic analysis of midbrain tissue from IPD patients and healthy controls

To date, transcriptomic studies in post-mortem human midbrain were based on bulk RNA sequencing technologies, hindering the study of the contribution of individual cell types to disease pathology. To overcome this challenge, we performed single-cell RNA sequencing of post-mortem human midbrain tissue of five IPD patients and six age-matched healthy controls to obtain an unbiased and global view of the cell type composition and cellular phenotypes of IPD. We identified all major cell types in the midbrain including different neuronal subtypes, glia and microvasculature cells and detected differentially expressed genes in each of those cell types. Moreover, we discovered an IPD-specific neuronal cluster characterized by the overexpression of CADPS2 and low TH expression, which suggests dysfunctional dopaminergic neurons. In addition, we observed a disease-specific upregulation of microglia and astrocytes further strengthening the role of neuroinflammation in PD (Smajic et al. Brain 2021). With additional analyses, we demonstrated the contribution of neuromelanin to the microglia activation in PD. This project was carried out within the framework of the ATTRACT project Model-IPD funded by the FNR.

To know more: Midbrain single-cell sequencing to understand Parkinson’s Disease (News published on 17 March 2022)

IPSC-derived microglia as a model to study inflammatory phenotypes in IPD

Neuroinflammation has been a hallmark of PD since the early 1980s. Many studies in human serum and cerebrospinal fluid samples have shown a significant upregulation of proinflammatory cytokines and chemokines in PD patients. This includes our own work, which identified increased levels of  circulating cell-free (ccf) mtDNA and the proinflammatory cytokine IL-6 in blood serum from PD patients with PRKN or PINK1 mutations (Borsche et al. Brain 2020). Inspired by these biomarker studies, we sought to investigate, if IPD inflammation can be modeled in iPSC-derived microglia. First, by analyzing multiple publicly available bulk RNAseq and snRNAseq datasets from post-mortem midbrain tissue, we discovered a significant upregulation of IL1B and IL10 in IPD patients compared to healthy controls. Furthermore, we identified microglia as being the predominant cell type involved in the propagation of inflammatory cascades. Second, to validate these findings in vitro, we generated iPSC-derived microglia. By treating our control and IPD iPSC-derived microglia with LPS, we indeed observed a higher expression of both IL1B and IL10 in IPD microglia. Moreover, this upregulation coincided with elevated levels of the NLRP3 inflammasome, indicative of stronger immune priming in IPD microglia (Badanjak et al. Front Cell Dev Biol 2021).

Alpha-synuclein is a key protein involved in idiopathic as well as genetic forms of PD, which has been associated with the release of inflammatory mediators and mitochondrial dysfunction. Using iPSC-derived microglia as well as neuron-glia co-cultures, we aim at studying the effect of alpha-synuclein on mitochondria modulation and NLRP3 inflammasome activation. This project is carried out within the framework of the CORE Junior project NeuroFlame funded by the FNR.

Markers and mechanisms of reduced penetrance in LRRK2 mutation carriers of PD

The manifestation of PD and the age of onset are not exclusively determined by the mutation identity, for instance in the LRRK2 gene. So far, only few factors have emerged, which define disease penetrance or constitute signs of advanced progression in LRRK2-PD. By contrast, in fibroblasts from manifesting and non-manifesting G2019S mutation carriers, we detected a correlation between mtDNA deletions and disease status (Ouzren et al. Ann Neurol 2019). Furthermore, cells from manifesting G2019S carriers had impaired complex I function as well as increased mitochondrial mass and mtDNA copy number, suggestive of impaired mitophagy. Finally, elevated expression of Nrf2 implicates ROS scavenging in the penetrance of LRRK2-PD (Delcambre et al. Front Neurol 2020). Thus, we now speculate that, in manifesting G2019S mutation carriers, increased LRRK2 kinase activity interferes with Nrf2 antioxidant signaling, which in turn (i) promotes mtDNA damage, (ii) mediates ccf-mtDNA release, and eventually (iii) triggers pro-inflammatory signaling. We are exploring this hypothesis in patient iPSC-derived neurons and microglia. Of note, iPSC-derived microglia from G2019S mutation carriers exhibit higher levels of pS1292 LRRK2, a known pathogenic form of phosphorylated LRRK2, and these levels are accompanied by significantly higher levels of pRab10, a known LRRK2 target. Our findings further strengthen the role of LRRK2 kinase activity in the pathogenesis of PD. We are currently investigating the therapeutic kinase inhibitor MLi-2, with or without inflammatory stimuli, to study the role of kinase activity in the inflammatory processes.

In addition, we investigated how environmental toxins may interfere with this molecular mechanism (Lüth et al. Front Aging Neurosci 2020). This project is part of the Research Unit “ProtectMove” (www.protect-move.de), which is co-funded by the FNR and the DFG.

Impact of PD-associated alpha-synuclein mutations on astrocytic metabolism

Recently, the neurocentric view of PD has been challenged with evidences that the disrupted interaction between neurons and glia contribute to the disease progression. Notably, there is a strong body of evidence indicating that astrocytic metabolism is pivotal to ensure proper neuronal function. In light of these findings, we aim to study the neuronal-astrocytic cooperation in astrocytes derived from patients harboring mutations in SNCA, with a special focus on metabolic alterations that could exacerbated neuronal pathology. Within the framework of the FNR-funded project CAMeSyn, we are collaborating with Dr Johannes Meiser (LIH) and Prof Rejko Krüger (LCSB, LIH). Furthermore, applying a differentiation protocol developed in the group of Prof Jari Koistinaho (University of Helsinki), we are generating pure non-activated astrocyte cultures from patients and controls, which are suitable for metabolomics studies, as well as cytokine profiling in the context of neuroinflammation.