INDIVIDUAL PROJECTS – WORK PACKAGE 3: NEUROIMMUNOLOGY

INTRODUCTION 

Parkinson’s disease (PD) is the 2nd most common neurodegenerative disease. Many of the key PD familial genes share a common role in regulating mitochondrial functions and dopaminergic (DA) neuronal survival in a cell-autonomous manner. However, the detailed molecular mechanisms underlying the pathogenesis of PD still largely remain elusive. With the recent discovery of a functional meningeal lymphatic system (Louveau et al., 2015), the under-appreciation of peripheral immune cells in the pathogenesis of neurodegenerative diseases is being increasingly noticed. Our unpublished dataset (Danileviciute et al, 2019, Biorxiv) show that a key PD familial gene is vital to modulating peripheral immune systems in both mice and patients with deficiency in that gene. We here plan to investigate the contribution of that specific gene via the adaptive immunity (AI) to the pathogenesis of PD, if successful, which might serve as a role model for the other PD-related genes and open a new window for the therapies of PD. 

AIM AND HYPOTHESIS 

As aforementioned, we reason that the observed dysregulated immune system in both KO mice and genetic PD patients might contribute to the pathogenesis of PD and consequently plan to investigate its potential causative role in the pathogenesis of PD. 

METHODS 

We will employ various conditional knockout lines in both immune subsets and DA neurons to distinguish the pathogenic contribution of the AI from the autonomous effects of DA neurons. In the mice models, we will analyse not only the cellularity and functions of various immune subsets, but also various PD-related neurological phenotypes, such as potential motor impairment, changes in TH+ cells among DA neurons and others. We will also utilise patient-derived iPSC-based models to investigate the effect of the gene of interest. 

INTRODUCTION 

Loss of function of DJ-1 is associated with autosomal recessive early-onset PD. DJ-1 is ubiquitously expressed in the CNS and acts as an intracellular redox sensor critical for neuroprotection (Cookson, 2010). In the absence of functional DJ-1, the nigrostriatal dopaminergic functions are dysfunctional (Goldberg et al., 2005). To what extent the loss of DJ-1 in vivo alters the function of microglia remains poorly understood. 

Our preliminary results indicate that microglia in DJ-1 KO mice display specific transcriptional programs that are different from WT mice with and without immune activation. 

AIM AND HYPOTHESIS 

We aim to characterise the transcriptional programs and immunological activity of microglia in DJ-1 deficient settings under homeostatic and inflammatory conditions. Based on our preliminary results, we hypothesise that the chronic activated/alerted microglia state associated to DJ-1 deficiency might contribute to the onset and progression of PD. 

METHODS 

We will take advantage of the DJ-1 KO mouse (Pham et al., 2010) and induced pluripotent stem cells (iPSCs) from DJ-1 deficient PD patients. We will FACS-isolate microglia from mouse brains (Sousa et al., 2018) and derive microglia from iPSCs (Haenseler and Rajendran, 2019) for transcriptional and functional analyses (e.g. mitochondrial phenotype in dopaminergic neurons, metabolic profiling and stress response) at single cell resolution. Notably, the use of iPSC‐derived microglia will pave the way to future drug screenings, which aim to restore microglia homeostatic phenotypes in PD patients. 

INTRODUCTION 

Recently, several genetic forms of Parkinson’s disease (PD) have been associated with neuroinflammatory processes (Grunewald et al., 2019). Mitochondria-derived damage-associated molecular patterns, such as reactive oxygen species (ROS) or circulating cell-free mitochondrial DNA (ccf-mtDNA), were shown to stimulate the cGAS–STING pathway (Sliter et al., 2018) or to directly initiate the formation of the NLRP3 inflammasome (Wei et al., 2019). Both processes mediate the maturation of inflammatory cytokines (Labzin et al., 2018). Our own preliminary analyses in serum from PINK1-PD and Parkin-PD patients provide evidence for a link between genotype, disease status, ccf-mtDNA levels and IL6 concentrations. Sequencing analyses of blood-derived mtDNA, additionally showed an association between intra-cellular mtDNA damage and cytokine release in these patients.  

Similarly, when assessing mtDNA integrity in non-manifesting and manifesting LRRK2-mutation carriers, we found an accumulation of somatic major arc deletions in the latter group (Ouzren et al., 2019). While this data supports a role for cGAS–STING and NLRP3 signaling in PD, evidence from induced pluripotent stem cell (iPSC)-derived neurons and glia is currently lacking. Moreover, it remains unclear if the activation of pro-inflammatory pathways is a phenomenon specific to PINK1, Parkin and LRRK2-associated PD. 

AIM AND HYPOTHESIS 

We hypothesise that inflammatory signaling connects mitochondrial dysfunction and neurodegeneration in PD and that this molecular link can be targeted with personalised anti-inflammatory treatment regimes. To address our hypothesis, we have defined the following aims for this PhD project: (i) to perform mitochondrial phenotyping and (ii) to assess cGAS–STING and the NLRP3 inflammasome signaling in microglia from carriers of mutations in Parkin, PINK1 and LRRK2. (iii) To extend this work to microglia from patients with mutations in the ROS scavenger DJ-1. (iv) Finally, we will use these newly established microglia models to screen for antiinflammatory compounds, which may interfere with cGAS–STING or NLRP3 signaling and reduce cytokine release. 

METHODS 

Experiments will include the generation of microglia-like cells from human iPSCs, transcriptomic and proteomic analyses of key inflammatory targets by RNAseq, Western blotting and ELISA. In addition, we will employ confocal microscopy and advanced automated image analysis techniques to determine cell activation and viability. The screening of FDA-approved antiinflammatory drugs will be performed on an automated high-content/high-throughput platform for iPSC-derived cellular models. 

INTRODUCTION 

Parkinson’s disease (PD) is, after Alzheimer’s disease, the second most prevalent neurodegenerative disorder. Although it is now widely accepted that PD is a complex “systems” disorder, the main characteristic of the disease is the degeneration of dopaminergic neurons in the substantia nigra of the midbrain. As a consequence of this degeneration the levels of the neurotransmitter, released in the Striatum, are decreasing. Additionally, a key neuropathological characteristic of PD is the appearance of the so called Lewy pathology. Clinically, PD is characterised by motor symptoms like rigidity, bradykinesia, tremor and postural instability. Often these motor symptoms are preceded by non-motor symptoms like hyposmia, constipation, sleep disturbances and depression. Microglia previously have been described to be critical for the PD pathogenesis. Microglia are the innate immune cells of the brain. They play a key role as the first line of defense of the CNS in case of infections but they also play a physiological role in synaptic homeostasis, maintenance, and functioning. In addition, they can have deleterious (pro-inflammatory phenotype) and/or positive effects (pro-repair phenotype) in different types of brain disorders. Recently protocols describing the derivation of microglia from human iPSCs have been described. 

AIM AND HYPOTHESIS 

We hypothesise that deregulated autophagy in PD patient specific microglia contributes to their deleterious activity during disease progression. Hence, we here aim at deriving microglia from iPSCs from patients from the NCER-PD cohort. Particularly patients with an immune phenotype (e.g. elevated levels of certain interleukins) would be interesting in this context. This project is part of a larger initiative within our lab. In order to analyse autophagy in them, iPSC derived microglia will be CRISPR/Cas9 engineered with fluorescent autophagy reports. Importantly, these reports are suitable for high content imaging and screening. Consequently, we will use this system to screen our available natural compound library for compounds targeting autophagy in PD specific microglia. Eventually, after identifying active compounds they will be validated in a next generation of PD patient specific brain organoids containing microglia. The project shall be in close collaboration with the NCER-PD initiative and patient selection will be based on clinical data from the cohort (collaboration with R. Krueger).  

The PhD candidate in this project, depending on the scientific background, shall contribute either to a) the generation of microglia and their phenotyping or to b) computational high content microscopy image analysis with artificial intelligence approaches. 

METHODS 

Cell Biology / Immunology background: 

(i) iPSC technology, (ii) differentiation into microglia and brain organoids, and (iii) high content imaging and compound screening. 

Computer science / Bioinformatics background: 

(i) image analysis / feature extraction, (ii) artificial intelligence approaches for image feature analysis (iii) correlation of imaging data with other datasets (e.g. transcriptomics or metablomics). 

INTRODUCTION 

The human gut microbiome is a complex ecosystem, which contributes essential functions to human physiology. Changes to the microbiome are associated with several chronic diseases characterised by inflammation, including Parkinson’s disease. Microbiome-derived effector molecules comprising nucleic acids, (poly)peptides and metabolites are present at high levels in the gut but have so far eluded systematic study. This gap in knowledge is limiting mechanistic understanding of the microbiome’s functional impact in Parkinson’s disease most notably how they are linked to higher levels of circulating inflammatory cytokines (TNF-alpha, IL-1, IL-6, IFN-gamma), chemokines (CXCL2, CXCL8, CXCL10) and inflammation markers [LPS binding protein, C-reactive protein]. 

AIM AND HYPOTHESIS 

The aim of the project is to resolve the microbiome-derived molecular complex in the gut and its impact on the human immune system in the context of Parkinson’s disease. The underlying hypothesis of the project is that gut microbiome-derived molecules stimulate pathways of innate and adaptive immunity, which trigger and/or contribute to Parkinson’s disease. 

METHODS 

The project will involve the generation, integration and analysis of quantitative, integrated multi-omic data of extracellular biomolecules from microbiome samples collected from healthy individuals and patients with newly diagnosed Parkinson’s disease. More specifically, the data will be integrated and analysed using a newly developed knowledge base (http://expobiome.lcsb.uni.lu). Using contextualised prior knowledge and machine learning methods, the project will identify microbial molecules associated with Parkinson’s disease-specific immunophenotypes.