Projects

Parkinson’s disease (PD) is characterized by α-synuclein-positive aggregates in the form of Lewy bodies and Lewy neurites. Point mutations, as well as duplications and triplications of the SNCA gene cause inherited forms of PD, and non-coding variants in influence disease risk and progression in sporadic patients, probably by modulating SNCA expression.

Epigenetics, i.e. the orchestration of transcriptional gene activity, regulated by multiple interacting mechanisms including, but not limited to gene promotor methylation, histone acetylation and the action microRNAs are a yet relatively poorly studied  but potentially important set of processes that may link genetic and environmental risks with endogenous processes like ageing in the causation of these complex disorders. The proposed project will use single-cell transcriptomics and epigenomic mapping of post-mortem material and different models, including neurons derived from patients with monogenic forms of PD, to understand the dysregulated networks leading to dopamine cell death.

Neurodegenerative diseases such as PS are characterized by numerous alterations in protein composition of the brain. However, the relevant brain cells are for obvious reasons practically  inaccessible during life. Recently it has been discovered, that neurons and other cells shed small membranous vesicles that contain small portions of their cytoplasm, called exosomes. Those exosomes can be recovered from the CSF, but also, to a smaller extent, from the blood. Those exosomes derived from neurons can be enriched using antibodies to proteins that are specifically expressed on neuronal cell membranes. In those, we are now looking for disease and stage specific protein profiles using highly sensitive assays.

Whole exome and whole genome sequencing (WES and WGS) on large cohorts of patients have found large numbers of potentially pathogenic genomic variants in patients but it is difficult to convincingly prove their causality using genetics alone. Similarly, Genome Wide Association Studies (GWAS) have identified large numbers of risk loci for disease but so far have not been investigated in detail to identify the actual causal variant. We aim to systematically investigate all identified variants from WES/WGS studies and all genes underneath the GWAS loci for their possible involvement in the pathogenesis of PD by using systematic high-throughput/high-content cellular screens using RNAi approaches. We will measure the effects of the genetic perturbations using a combination of microscopic readouts and RNAseq thus allowing to evaluate the molecular effects of each variant within a neuronal cell at high resolution and in relation to the processes that are disturbed in the disease.

Although symptomatic treatment has been successful for PD, no therapy exists that halts or slows down the neurodegenerative process. We hypothesise that clinical trials have consistently failed for three main reasons. First, selection of potential drug targets is rarely based on convincing biological evidence. Second, the disease process begins decades before clinical symptoms are observed and clinical trials on patients are therefore likely too late to reverse the neurodegeneration. Third, participants have been selected largely ignoring their underlying disease biology, phenotypic variation and their genetic risk profile, resulting in a very heterogeneous population of cases with very different progression of disease.

Therefore we aim to develop a novel concept for disease-mechanism based disease onset and progression prediction and subsequent target discovery and patient stratification for PD. Our goals are to generate novel targets based on biological evidence with matching precision cohorts for clinical trials that will allow for personalised therapeutic interventions based on genetic and genomic risk profiles and clinical subtypes.

To reach our goal, we will use a systems biology approach by generating multi-dimensional clinical, genetic/genomic and biological risk and progression profiles for our patients and at risk individuals and integrate these data in gene expression models for target discovery and disease prediction and progression models for patient stratification.

This project is funded in part by  the Michael J Fox PATH to PD program that aims to holistically investigate known risk and causal factors toward discovery of common framework underlying onset and progression of Parkinson's disease (http://www.foundinpd.org/). We  will grow Dopaminergic neurons from 100 induced pluripotent stem cell lines  and use advanced "omics" techniques (e.g., genomics, proteomics, metabolomics) to map how various genetic changes lead to cellular and molecular changes associated with PD.

This work is complemented by the EraCoSysMed funded project, PD-Strat that aims to generate multi-dimensional clinical, genetic/genomic and biological risk and progression profiles in our well-characterised clinical cohorts and use these risk profiles in cellular models to identify new targets based on biological evidence and generate precision cohorts suitable for clinical trials on well-defined subsets of patients.

As mutations within the protein kinase LRRK2 (Leucine-rich repeat kinase 2) are involved in the pathophysiology of familial, as well as sporadic, PD and lead to increased kinase activity, it is considered as a promising drug target for disease treatment. Besides a kinase domain, the multi-domain protein LRRK2 possesses a G-domain which is structural similar to Ras-like small G-proteins but with biochemical unique features. LRRK2 has recently been shown to be involved in the regulation of vesicular trafficking by phosphorylating a specific subset of Rab proteins. Current research suggests that LRRK2 forms active complexes at specific endo-membranes in the post-Golgi compartment and PD-variants of LRRK2 have been demonstrated to impair autophagy as well as mitochondrial functions. To support the development of specific and safe drugs for LRRK2-PD, we are currently working on a comprehensive biochemical analysis combined with interactomics and structural biology to identify the activation mechanism of LRRK2 at a molecular and atomic level.

The overall goal of our research is to understand whether and how the interaction between genetic risk, age related metabolic decline and immune dysfunction contribute to the development and progression of neurodegenerative diseases with a particular focus on Parkinson’s disease. Inflammatory genes have been associated with several neurodegenerative diseases, including Parkinson's disease. Our aim is to understand, at the cellular and molecular level, how disease-associated genetic variants affect immune cell metabolism, and how immune responses within or outside the brain contribute to neurodegeneration. We do that by combining patient neurons derived from induced pluripotent stem cells and CRISPR-Cas9 genome editing techniques.

Heterozygous mutations in the GBA gene represent the most common genetic risk factor for PD. We built-up a large cohort of PD patients carrying a GBA mutation (PDGBA). Our own findings of PDGBA patients to present with an earlier age at onset, more prominent non-motor symptoms (cognitive impairment, neuropsychiatric disturbances and autonomic dysfunction) and a more rapid disease progression have been replicated in several other studies. Next to extensive clinical data, a huge battery of biomaterials (blood, CSF, fibroblasts) have been collected allowing in-depth biomarker analysis of the lysosomal pathway in relation to clinical phenotypes. Importantly, there is a more general relationship between lysosomal insufficiency and PD. This allows pathway-specific analyses on pathophysiology and treatment options.

 α-Synuclein is one of the top-most genetic risk factors for PD and the protein is the major building block of Lewy bodies, the neuropathological hallmarks in the brain of PD patients. We are using transgenic mice expressing human mutant A30P α-synuclein under the control of a Thy1 promoter, which recapitulate human α-synucleinopathy down to the ultrastructural level. Cognitive behavior of (Thy1)-h[A30P]αSYN mice is impaired in an age-dependent manner, most likely due to development of neuropathology and neuronal dysfunction within the amygdala circuitry. Moreover, old transgenic mice ultimately die of locomotor deterioration, caused by brain stem and spinal motoneuron pathology. The age of onset of this terminal phenotype is accelerated by high fat diet-induced obesity (Rotermund et al. 2014). We are further investigating epigenetic mechanisms influenced by α-synuclein in cell culture and in vivo (Sugeno et al. 2016).

Most of the familial PD cases are caused by recessive mutations in the PARK2/PARKIN gene, which may also be a genetic risk factor for sporadic PD. The PARKIN gene product functions as an E3 ubiquitin protein ligase for a variety of unrelated substrate proteins. We investigate the role of parkin in the autophagic degradation of damaged mitochondria. Parkin is recruited to experimentally depolarized mitochondria in a PINK1-dependent manner, which is differentially affected by PD mutations in both genes (Geisler et al. 2010). Parkin ubiquitinylates a distinct set of mitochondrial outer membrane protein in a highly complex manner. We are further investigating the complex regulation of PINK1/parkin-regulated mitophagy, also using high content imaging.