The Defeat Parkinson’s Platform Tübingen

Parkinson’s disease (PD) is one of the most devastating neurodegenerative conditions. Although dopamine replacement treatment or deep brain stimulation can give remarkable temporary benefit to many patients, there is still no cure and it is not yet possible to significantly slow the progression of the disease.

However, in recent years we have seen a remarkable progress in the understanding of the genetic, molecular and cellular underpinnings of the disorder, and new treatments may be within reach in the not too distant future.

Supported by a major private donation and research grants from the Michael J. Fox Foundation (MJFF) and EU worth more than 2,5 Million Euro, the Hertie-Institute for Clinical Brain Research and the German Center for Neurodegenerative diseases (DZNE) in Tübingen have established the Defeat Parkinson’s Research Platform. Clinical and pre-clinical researchers will be joining forces to use cutting edge molecular, genetic, cell biologic and computational technologies to better understand the complex mechanisms underlying the disorder and to develop novel biomarkers and treatments.

Mission statement

In the Defeat Parkinson’s Research Platform we will work together in an interdisciplinary team to explore how genetic variability leads to alterations in cellular and molecular processes that define disease risk and progression. To achieve this goal, we will join forces with our international collaborators (http://pdgenetics.org/) to investigate the entire genomic variation in large groups of affected and at-risk individuals with next-generation sequencing technologies and correlate this data with transcriptome and epigenome information to build gene expression models. We will then explore how these changes alter cellular processes by high content/high throughput analysis, even at the single cell level, use novel computational approaches to integrate these findings in complex models. With our expert knowledge on disease modeling in induced pluripotent stem cells (iPSc) and animal models we will then investigate selected genes in depth and bring our knowledge back to our patients to discover and validate novel biomarkers and treatments.

Through the enthusiasm of our teams, with the input of numerous collaborators and the help of generous funders we are confident that we will make a difference to PD research and find new treatments that better the lives of our patients and eventually will lead to a cure or even prevention of PD.

  • Principle Investigators

    Thomas Gasser is Professor of Neurology and director of the Department for Neurodegenerative Diseases at the HIH and the University Hospital for Neurology and heads the Parkinson’s Genetics group at the HIH and the DZNE Tübingen. He has led one of the teams discovering the LRRK2 gene as the most common cause for autosomal dominantly inherited PD and has, together with collaborators, pioneered GWA studies in PD.

    Peter Heutink is head of the Genome Biology of Neurodegeneration group and speaker of the DZNE-Tubingen. His team has identified genes causing inherited PD and risk loci for sporadic PD. His current interest focusses on the investigation of the molecular effects of the large number of risk genes identified for both familial and sporadic PD using high throughput genomic, bioinformatic and cellomic methods in induced Pluripotent Stem (iPS) cell models.

    Philipp Kahle is head of the Functional Neurogenetics group at the HIH/DZNE Tübingen. Research aims at the understanding of neuropathological and epigenetics mechanisms of a-synuclein as well as mitochondrial quality control mechanisms regulated by recessive PD genes. More information

    Michela Deleidi is a Helmholtz group leader at the DZNE-Tübingen. Her team works on the molecular mechanisms of neurodegeneration, with a special emphasis on the role of inflammation in Parkinson's disease. More information 

    Kathrin Brockmann heads the Parkinson’s disease outpatient unit at the Department of Neurology and is affiliated with the Integrated Clinical and Research Unit (ICRU) of the DZNE Tübingen. Her team focuses on patient stratification according to imaging patterns, genetic architecture and biomarkers as one of the major prerequisites to develop  pathway-specific and milestone-related therapies.

    Inga Liepelt is a neuropsychologist. As head of the Integrated Clinical and Research Unit (ICRU) of the DZNE Tübingen, she is responsible for a seemless workflow in numerous clinical trials. She also runs studies on cognitive and behavioural interventions in PD.

    Julia Fitzgerald is a junior group leader at The Hertie-Institute for Clinical Brain Research, Tübingen working on the biological processes underlying neuronal cell death in Parkinson’s disease. Using genetic models and patient derived cells, the group focus on biochemical signaling and have a long standing specialism in mitochondria and the role of mitochondrial proteins in Parkinson’s disease.

    Christian Johannes Gloeckner is group leader at the DZNE-Tübingen. His team is working on the functional analysis of the PD-associated proteins. The current focus of the work is on the functional analysis of LRRK2 by systematic mapping of protein-interaction networks, by biochemical methods as well as by structural modelling. More information

  • Facilities and Ressources

    Clinical setting

    The clinical Department of Neurodegenerative Diseases runs largest Parkinson’s outpatient clinic in southern Germany, specialized in early and differential diagnosis of the disorder. Patients with severe disease are treated on the departments inpatient ward. This allows physicians and researchers to follow large longitudinal cohorts and to collect clinical data and biospecimens with the goal to find new biomarkers and factors that modify the course of the disease. Together with the DZNE Tuebingen, an Integrated Care and Research Unit (ICRU) with a trained staff of neurospychologists, study nurses and technicians has been created to facilitate observational and inverventional clinical trials.

    Probands of DZNE-studies are now also seen in the new study center at the DZNE building.

    Cohorts

    Since more than 10 years, physicians at the clinical Department of Neurodegenerative diseases have systematically followed patients with Parkinson’s disease and persons at risk for PD and other neurodegenerative diseases. Many of these individuals have donated blood, DNA and other biomaterials to our BioBank. Careful documentation of clinical signs and symptoms allows to correlate genetic risks and biomarker profiles to the evolution of the disease, giving important insight into the evolution of the disease. Care is taken to comply to data safety regulations of the highest standards.

    BioBank

    Together with the DZNE, the center of Neurology in Tübingen jointly runs one of the largest BioBanks for Parkinson’s disease worldwide. For example, more than 4000 DNA samples, 2000 CSF and blood samples from patients with different parkinsonian syndromes have been collected under the highest quality standards and are available for researchers. In many of them, longitudinal data on disease evolution are available, making these samples particularly valuable. Other body fluids and fibroblasts from patients and controls are also banked. Importantly, through the clinical department, new material can be obtained.

    Genetic pipelines

    Many of our patients have consented to be screened for genetic variants that may cause or influence the course of the disease. Together with many collaborators world-wide, we are therefore able to identify the causative genes in familial cases and to explore the contribution of genetic factors the more common sporadic forms of PD. Today we are already running the first clinical trials in patients with certain gene mutations trying to the disturbed cellular pathways. Eventually we expect that this research will enable us to predict the development and progression of the disease and its various symptoms and to provide earlier and more targeted treatments with ever greater accuracy.

    Human induced pluripotent stem cells

    The nobelprize-winning discovery that differentiated cells can be turned into induced pluripotent stemc cells (IPSCs) and then made to differentiate into almost any cell type has revolutionized brain research, because it means that brain cells from patients, that previously had been practically unaccessible during life, can now be studied in a dish. In Tübingen, we are extensively using this technology, to the extent that we can study thousands of individual cultures in a highly automated fashion (cellomics).

    Genomic resources

    The DZNE Tubingen, the HIH and the clinical neurology department have created a state of the art platform for genomic research, using transcriptomics, epigenetics, proteomics and high-throughput cellomics to analyze cells from a large number of well characterized patients. A team of data scientists explores the data and collaborates with scientists from other disciplines to connect and integrate the data with non-genomic datasets. The integration of genomic data with microscopy and/or phenotypic data will help to generate new hypothesis and open new avenues of research. We are embedded in an excellent environment at Tübingen University, with its Quantitative Biology Platform QBiC. In addition, Tübingen has been selected as one of only three “next generation sequencing hubs” by the DFG, providing  unparalleled capacities for sequencing and data analysis.

    Collaborations

    Researchers and physicians of the Defeat Parkinson's Platform are active and often leading members of many international research networks.

    • the International Parkinson's Disease Genomics Consortium [IPDGC],
    • the MJFF-funded PPMI and P-PPMI Study
    • the MJFF LRRK2 biological consortium,
    • the Centre-PD consortium (Luxemburg, Oxford, Tübingen),
    • DecipherPD consortium on epigenomics of Parkinson’s disease

    Funding

    The Defeat Parkinson’s Platform is supported by institutional funds from the Charitable Hertie Foundation, the University of Tübingen, the German Center for Neurodegenerative diseases (DZNE) and by project funding from the German research Council (DFG), the German ministry for science and research (BMBF), the European Commission (EC), the Michael J Fox Foundation for Parkinson’s Research (MJFF), iMed—the Helmholtz Initiative on Personalized Medicine, and private donors.

    Funded projects:

  • Projects

    Single cell epigenomics of PD (Gasser)

    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.

    Biomarker signatures in brain derived exosomes (Gasser)

    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.

    Systematic functional characterization of genetic risk factors (Peter Heutink)

    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.

    Multi-omics stratification of PD patients for personalised interventions. (Peter Heutink)

    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.

    Functional analysis of LRRK2 (Johannes Gloeckner)

    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.

    Inflammation and immune cell metabolism in Parkinson's disease (Michela Deleidi)

    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.

    Pathways associated with PD and phenotypic variability: lysosomal Dysfunction (Kathrin Brockmann)

    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.

    Neuropathological and Epigenetic Mechanisms of α-Synucleinopathy (Philipp Kahle)

     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).

    Regulation of PINK1/Parkin-mediated Mitophagy (Julia Fitzgerald)

    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.

  • Publications
    • Brockmann K, Lerche S, Dilger SS, Stirnkorb JG, Apel A, Hauser AK, Liepelt-Scarfone I, Berg D, Gasser T, Schulte C, Maetzler W: SNPs in Abeta clearance proteins: Lower CSF Abeta1-42 levels and earlier onset of dementia in PD. Neurology 2017, 89:2335-2340.
    • Brockmann K, Hilker R, Pilatus U, Baudrexel S, Srulijes K, Magerkurth J, Hauser AK, Schulte C, Csoti I, Merten CD, et al: GBA-associated PD. Neurodegeneration, altered membrane metabolism, and lack of energy failure. Neurology 2012, 79:213-220.
    • Brockmann K, Srulijes K, Hauser AK, Schulte C, Csoti I, Gasser T, Berg D: GBA-associated PD presents with nonmotor characteristics. Neurology 2011, 77:276-280.
    • Deleidi M, Jäggle M and Rubino R. "Immune ageing, dysmetabolism and inflammation in neurological diseases". Frontiers in Neuroscience. Front Neurosci. 2015 Jun 3;9:172. doi: 10.3389/fnins.2015.00172.
    • Geisler, S., Holmström, K. M., Skujat, D., Fiesel, F. C., Rothfuss, O. C., Kahle, P. J., and Springer, W. (2010). PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat. Cell Biol. 12, 119-131
    • Gloeckner CJ, Kinkl N, Schumacher A, Braun RJ, O'Neill E, Meitinger T, Kolch W, Prokisch H, Ueffing M. The Parkinson disease causing LRRK2 mutation I2020T is associated with increased kinase activity. Human molecular genetics. 2006;15(2):223-32. doi 10.1093/hmg/ddi439
    • Guaitoli G, Raimondi F, Gilsbach BK, Gomez-Llorente Y, Deyaert E, Renzi F, Li X, Schaffner A, Jagtap PK, Boldt K, von Zweydorf F, Gotthardt K, Lorimer DD, Yue Z, Burgin A, Janjic N, Sattler M, Versees W, Ueffing M, Ubarretxena-Belandia I, Kortholt A, Gloeckner CJ. Structural model of the dimeric Parkinson's protein LRRK2 reveals a compact architecture involving distant interdomain contacts. Proceedings of the National Academy of Sciences of the United States of America. 2016;113(30):E4357-66. doi 10.1073/pnas.1523708113
    • Jansen IE, Ye H, Heetveld S, Lechler MC, Michels H, Seinstra RI, Lubbe SJ, Drouet V, Lesage S, Majounie E, Gibbs JR, Nalls MA, Ryten M, Botia JA, Vandrovcova J, Simon-Sanchez J, Castillo-Lizardo M, Rizzu P, Blauwendraat C, Chouhan AK, Li Y, Yogi P, Amin N, van Duijn CM; International Parkinson Disease Consortium,  Heutink P. Discovery and functional prioritization of Parkinson's disease candidate genes from large-scale whole exome sequencing. Genome Biol. 2017 Jan 30;18(1):22. doi: 10.1186/s13059-017-1147-9.
    • Khurana V, Peng J, Chung CY, Auluck PK, Fanning S, Tardiff DF, Bartels T, Koeva M, Eichhorn SW, Benyamini H, Lou Y, Nutter-Upham A, Baru V, Freyzon Y, Tuncbag N, Costanzo M, San Luis BJ, Schöndorf DC, Barrasa MI, Ehsani S, Sanjana N, Zhong Q, Gasser T, Bartel DP, Vidal M, Deleidi M, Boone C, Fraenkel E, Berger B, Lindquist S. "Genome-scale networks link neurodegenerative disease genes to alpha-synuclein through specific molecular pathways". Cell Syst. 2017 Jan 25. pii: S2405-4712(16)30445-8.  doi: 10.1016/j.cels.2016.12.011.
    • Mittal S, Bjørnevik K, Im DS, Flierl A, Dong X, Locascio JJ, Abo KM, Long E, Jin M, Xu B, Xiang YK, Rochet JC, Engeland A, Rizzu P, Heutink P, Bartels T, Selkoe DJ, Caldarone BJ, Glicksman MA, Khurana V, Schüle B, Park DS, Riise T, Scherzer CR. β2-Adrenoreceptor is a regulator of the α-synuclein gene driving risk of Parkinson's disease. Science. 2017 Sep 1;357(6354):891-898.
    • Piccoli G, Onofri F, Cirnaru MD, Kaiser CJ, Jagtap P, Kastenmuller A, Pischedda F, Marte A, von Zweydorf F, Vogt A, Giesert F, Pan L, Antonucci F, Kiel C, Zhang M, Weinkauf S, Sattler M, Sala C, Matteoli M, Ueffing M, Gloeckner CJ. Leucine-rich repeat kinase 2 binds to neuronal vesicles through protein interactions mediated by its C-terminal WD40 domain. Mol Cell Biol. 2014;34(12):2147-61. doi 10.1128/MCB.00914-13
    • Robak LA, Jansen IE, van Rooij J, Uitterlinden AG, Kraaij R, Jankovic J; International Parkinson’s Disease Genomics Consortium (IPDGC), Heutink P, Shulman JM; IPDGC Consortium members; International Parkinson’s Disease Genomics Consortium (IPDGC). Excessive burden of lysosomal storage disorder gene variants in Parkinson's disease. Brain. 2017 Nov 13. doi: 10.1093/brain/awx285. [Epub ahead of print]
    • Rotermund, C., Truckenmüller, F. M., Schell, H., and Kahle, P. J. (2014) Diet-induced obesity accelerates the onset of terminal phenotypes in a-synuclein transgenic mice. J. Neurochem. 131, 848-858
    • Schöndorf DC, Aureli M, McAllister FE, Hindley CJ, Mayer F, Schmid B, Sardi SP, Valsecchi M, Hoffmann S, Schwarz LK, Hedrich U, Berg D, Shihabuddin LS, Hu J, Pruszak J, Gygi SP, Sonnino S, Gasser T*, Deleidi M *. "iPSC-derived neurons from GBA1-associated Parkinson's disease patients show autophagic defects and impaired calcium homeostasis". Nat Commun. 2014 Jun 6;5:4028. doi: 10.1038/ncomms5028.
    • Simon-Sanchez J., Schulte C., Bras J. M., Sharma M., Gibbs J. R., Berg D., Paisan-Ruiz C., Lichtner P., Scholz S. W., Hernandez D. G., Kruger R., Federoff M., Klein C., Goate A., Perlmutter J., Bonin M., Nalls M. A., Illig T., Gieger C., Houlden H., Steffens M., Okun M. S., Racette B. A., Cookson M. R., Foote K. D., Fernandez H. H., Traynor B. J., Schreiber S., Arepalli S., Zonozi R., Gwinn K., van der Brug M., Lopez G., Chanock S. J., Schatzkin A., Park Y., Hollenbeck A., Gao J., Huang X., Wood N. W., Lorenz D., Deuschl G., Chen H., Riess O., Hardy J. A., Singleton A. B., Gasser T. Genome-wide association study reveals genetic risk underlying Parkinson's disease. Nat Genet. 2009;41(12):1308-12.
    • Sugeno N, Jäckel S, Voigt A, Wassouf Z, Schulze-Hentrich J, Kahle PJ. (2016) α-Synuclein enhances histone H3 lysine-9 dimethylation and H3K9me2-dependent transcriptional responses. Sci Rep. 6, 36328
    • Zimprich A., Biskup S., Leitner P., Lichtner P., Farrer M., Lincoln S., Kachergus J., Hulihan M., Uitti R. J., Calne D. B., Stoessl A. J., Pfeiffer R. F., Patenge N., Carbajal I. C., Vieregge P., Asmus F., Muller-Myhsok B., Dickson D. W., Meitinger T., Strom T. M., Wszolek Z. K., Gasser T. Mutations in LRRK2 Cause Autosomal-Dominant Parkinsonism with Pleomorphic Pathology. Neuron. 2004;44(4):601-7.

Donate

If you would like to support our work financially, you will find further information here.

Info-Hotline

Thursdays 1:30-4:30 pm

Patients +49 800-7799001

(free of charge)

Professionals +49 180-779900

(9 Cent/Min. German landline, mobile and out of Germany possibly more expensive)