Research projects

The relevance of Miro1 for mitochondrial function and dynamics in PD

Recent studies found a loss of function of Parkinson’s Disease (PD)-associated proteins PINK1, Parkin and DJ-1, causing impairment of mitochondrial quality control and subsequent loss of neurons. Mitochondrial quality control requires a well-regulated interplay of the mitochondrial Rho GTPase Miro1 with the PINK1/Parkin pathway. The aim of this project is to characterize the influence of Miro1 in pathways relevant to PD, such as mitochondrial energy metabolism, calcium homeostasis, degradation and transport. For this project, we have unique access to novel PD-associated mutations in the RHOT1 gene, encoding Miro1 protein, in order to investigate the consequence of such mutations on mitochondrial and neuronal homeostasis. First results from patient-derived fibroblasts show that mutations of RHOT1 lead to impaired cellular calcium homeostasis and increased mitochondrial turnover, making cells more prone to stress. Currently, we investigate the consequence of the observed mitochondrial phenotypes for neuronal function, using transgenic mice and iPSC-derived neurons.

GBA mutation as a modifier of familial Parkinson’s disease

Mutations in more than 20 loci have been identified as being causative for genetic forms of PD. Amongst them mutations in LRRK2 represent the most-common cause of autosomal dominant PD and mutations in parkin are the most-common mutations found in recessive inheritance familial PD. Nevertheless, a heterogeneity in the penetrance of the pathology, the phenotype or the age of onset of patients carrying these mutations leads to the search of associated factors that could influence the development of the disease. Mutations in the GBA gene, encoding the beta-glucocerebrosidase, are an important and common risk factor for both familial and sporadic PD. Carriers of one mutated allele of GBA have a 5-fold increased risk to develop PD and are more likely to progress to dementia, develop axial symptoms and have a slightly earlier age of onset compared to non-carrier PD patients. Underlying these pathological changes, modifications of metabolic pathways and cellular activity lead to neuronal impairment in patients.

To explore the effects of the GBA mutations, we are using cellular models derived from fibroblasts of two PD patients, harbouring mutations in GBA and mutations in another  gene causing of the disease. These fibroblasts, reprogrammed into induced pluripotent stem cells, can be further differentiated to small neuronal precursor cells to finally generate midbrain-specific dopaminergic neurons. Phenotyping these cells in terms of protein expression, lysosomal activity or mitochondrial morphology and function will allow us to understand the molecular basis of GBA effects in PD.

Exploring the effects of GBA mutations in the context of monogenic PD with well-known mutations will open new perspectives in the comprehension of combined/additive effects of different genes in the same individual.

DJ-1 and molecular pathways

Familial forms of Parkinson’s disease (PD) offer the opportunity to generate human cell models based on induced pluripotent stem cell (iPSC) technology to study the pathophysiology of the disease. These models are used to identify molecular pathways involved in PD and to gain general insights not only into familial but also into idiopathic PD. One of the causes of familial PD is homozygous loss-of-function mutations of DJ-1. DJ-1, a protein encoded by the gene PARK7, has broad biological functions including effects on mitochondrial and lysosomal homeostasis (Krebiehl et al.). We have previously shown that fibroblasts obtained from PD patients carrying the homozygous mutation c.192G>C in the DJ-1 gene display a phenotype of impaired mitochondrial respiration, increased intra-mitochondrial reactive oxygen species, reduced basal autophagy and the accumulation of defective mitochondria.
 
In order to study the effect of DJ-1 loss of function on PD target cells, midbrain-specific dopaminergic (mDA) neurons, we have generated iPSC from these fibroblasts. Using a new approach, we differentiate iPSC first into small molecule neuronal precursor cells (smNPC) that can be expanded indefinitely and, if needed, differentiated into mDA neurons within three weeks. Using pairs of disease-specific and isogenic and non-isogenic healthy control iPSC we’re identifying cellular phenotypes in mDA neurons that can be used as read out for small chemical compound library screens. Three commercially available compound libraries will be screened in the first round to identify compounds that rescue PD phenotypes in our DJ-1 model.  Therefore, we’re currently establishing a full automation of our cell culture including high content imaging to perform high through put screening in our lab.

Results have been published in Science Translational Medicine in September 2020.
To know more, read the press release and the full article. 

Pathological role of α-synuclein in Parkinson’s disease

Following the discovery that SNCA encodes α-synuclein (Polymeropoulos et al., 1997) in an A53T autosomal dominant form of inheritance in a familial case of PD, a further five other point mutations have been identified in families with hereditary PD: A30P (Kruger et al., 1998), E46K (Zarranz et al., 2004), H50Q, (Appel-Cresswell et al., 2013), G51D (Lesage et al., 2013) and A53E (Pasanen et al., 2014).   Furthermore, duplications and triplications of SNCA have been identified leading to the clinical manifestation of the disease, with the triplication of SNCA leading to an earlier disease onset and increased diseased severity.  Several Genome Wide Association Study (GWAS) have also implicated the variability at the SNCA locus as a major risk factor in idiopathic PD (Simon-Sanchez et al., 2009).  The oligomerisation of the monomeric α-synuclein into toxic aggregates of amyloid-like fibrils is one of the main pathogenic features of α-synuclein, which is a major constituent of LB (Spillantini et al., 1998).

At the Krüger lab, we are currently working with patient-derived cells containing the SNCA point mutation’s in p.A30P and p.A53T and also the duplication and triplication of the SNCA locus.  We have generated single-cell isogenic clones correcting these disease-causing point mutations and have introduced these mutations into age- and gender-matched controls.  The focus of our research involves the differentiation of the SNCA cell lines and controls into ventral midbrain dopaminergic neurons (vmDANs) and astrocytes from a renewal population of neuronal precursor cells (NPCs).  Our current work involves the investigation of phenotypes of these SNCA cell lines, specifically exploring the link between α-synuclein and mitochondrial function.  We have also generated a robust model using mDANs for high-throughput toxicity screening (HTS), we are currently working with collaborators for drug discovery and drug repurposing, and are open for future collaboration in this regard.

Some of the key analytical tools that enable us to asses these PD phenotypes include Spinning Disk and STED confocal microscopy, Multi-Electrode Arrays (MEAs) and an Extracellular Flux Analyser (Seahorse).  We also use important analytical techniques such as qPCR, immunocytochemistry, flow cytometry and protein immunoblotting to quantify and qualify functionality and specific vulnerability of these specific patient-derived mDANs.

Figure: Patient-derived PD mDANs.  Cells are triple stained with the nuclear marker Hoechst; the midbrain marker FoxA2 and Tyrosine Hydroxylase (TH): the rate-limiting enzyme that is responsible for catalysing L-tyrosine to L-Dopa.

Cellular endophenotypes of VPS35 mutation in Parkinson’s disease

The VPS35 D620N mutation has been identified as an autosomal-dominant cause of familial PD. The VPS35 protein is a component of the retromer complex and is implicated in the sorting and trafficking of various proteins from endosomes to the trans-Golgi network. Recent studies have revealed the role of VPS35 in another trafficking pathway from mitochondria to peroxisomes through mitochondria-derived vesicles. The D620N mutation is thought to lead to impaired trafficking of proteins, causing dysfunctions in mitochondria and autophagy.

The aim of this project is to study the phenotype of midbrain dopaminergic neurons (mDA) derived from patient’s fibroblasts carrying the VPS35 D620N mutation. We use live cell imaging techniques, biochemical and functional assays to study the impact of the VPS35 D620N mutation on mitochondria and autophagy.

Disease Modifiers in LRRK2 Parkinson’s Disease

Parkinson’s disease (PD) is the most frequent neurodegenerative movement disorder. While several genes causative for familial forms of PD as well as risks factors have already been identified, it is still unclear how patient’s genomes shape their predisposition to develop PD. For example, despite sharing the same mutation in the LRRK2 gene – the most common cause of late-onset familial PD – carriers display broad variation in severity of symptoms as well as the time of disease onset. To address this observed and unexplained variation, the LCSB takes part in an international effort, collaborating with partners in several European countries, to study families sharing both a history of PD and a G2019S mutation in the LRRK2 gene. By comparing clinical data and genetic profiles of patients a list of susceptibility factors has been generated (ongoing collaboration with Dr. Enrico Glaab and Dr. Patrick May at the LCSB). We are using iPSC based models to generate dopaminergic neurons derived from PD patients and healthy controls to investigate the effects of these susceptibility factors on PD related phenotypes in vitro.

Functional analysis of Parkinson’s disease-associated genes

Parkinson´s disease is most likely caused by a complex interplay between genetic and environmental factors. Many of the genes causing familial forms of PD have been identified, however, the molecular and cellular mechanisms leading to the specific hallmarks of this neurodegenerative disease remain unknown. As mitochondrial dysfunction is a common feature of both familial and idiopathic forms of PD, we carry out a functional analysis of genes underlying mitochondrial dysfunction in PD. Single and combinatorial gene perturbations are applied via gene knockdown and gene overexpression methods in neuronal cell culture models. Subsequently, a number of physiological and pathophysiological processes are analysed, i.e. the mitochondrial membrane potential and turnover, NADH turnover or ROS production. For this purpose, we perform live-cell imaging experiments combined with an automated image analysis pipeline to extract a wide range of features that characterise cellular and organelle function and help to potentially stratify PD into specific mechanism-based subtypes.

National Centre of Excellence in Research on Parkinson's Disease (NCER-PD) 

PD patients and healthy subjects are recruited in Luxembourg and the Greater Region. The aim is to improve diagnosis and stratification of PD by developing novel disease biomarkers. This project is funded by the Luxembourg National Research Fund (FNR) anf further information can be found at page 20 flagship programmes.

Interdisciplinary Collaboration: Treatment of gait disturbances in Parkinson’s disease

The team participates in a multicentre clinical study on the implementation of novel deep brain stimulation to treat therapy-resistant gait-freezing in advanced PD patients. A novel concept targeting the substantia nigra pars reticulata in the brain is used in a clinical study, supported by Medtronic, comprises 10 centres in Germany and Luxembourg.

ParkinsonNet-Luxembourg and Programme Démence Prévention 

Our group develops innovative patient care concepts for PD and other conditions. We coordinate ParkinsonNet-Luxembourg, which brings healthcare professionals together and facilitates Parkinson-specific specialisation, interdisciplinary collaboration and exchange of knowledge, allowing that every PD-patient in Luxembourg receives the best possible care. In addition, we lead the Programme Démence Prévention (pdp). The aim of pdp is to implement a programme launched by the Luxembourgish Ministry of Health allowing, by the means of a personalized lifestyle intervention, to prevent, or at least delay, the development of dementia in a target population, defined by a mild cognitive impairment.