Nijmegen Centre for Mitochondrial Disorders (NCMD) 

The Nijmegen Centre for Mitochondrial Disorders is a full-facility international reference centre for clinical care, biochemical, pathological and molecular diagnostics and research of patients suspected for or with established disturbances of the mitochondrial energy metabolism. The mitochondrial research is acknowledged in the Nijmegen Center for Molecular Life Sciences theme Metabolic Genomics and the main research program Metabolic and Genetic Disorders of the UMCN.

The main topic of our research concerns the oxidative phosphorylation (OXPHOS) system in health and disease with the final aim to develop new treatment strategies for genetic disorders of the system in man. To improve advancement of therapeutic approaches including development lead components NCMD has started strategic partnership with the expert with the company Khondrion (http://www.khondrion.nl/ ) founded by professor Smeitink.

The NCMD has an extensive international network and we are able to offer students the possibility to do a traineeship abroad.

Student projects may involve a traineeship in the following research topics:

(I) Biogenesis of human complex I; proteins involved, crucial steps, regulation and role in disease.
(Dr. Leo Nijtmans, Dept. Pediatrics UMCN; Tel: 024-3610938; )

Research interests include the biogenesis of mitochondria and in particular the complexes involved in the process of OXPHOS, with a specific focus on one of the largest multi-protein complexes that are known in nature: Complex I (CI). The assembly of this giant enzyme is very intricate in view of the fact that it consists of at least 44 subunits which are encoded either by the nuclear or mitochondrial DNA.

Research questions investigated comprise: what is the sequence of assembly of subunits of CI? What are the crucial steps in the assembly? How is the biogenesis of complex I regulated? How is assembly affected in CI deficient patients? Answers to these research questions provide insight in the molecular mechanisms leading to mitochondrial disorders.

More specifically, in our research we mainly focus on:

• The role of (candidate) assembly chaperones and their possible binding partners.
• Effects of specific knockdown of CI subunits by RNA interference on the assembly of CI.
• Assembly of CI in living cells by using fluorescent tags.
• CI assembly defects in patient cells.

During a traineeship students are able to get experienced with a divers spectrum of techniques:

• RNA interference
• 1D Blue-Native PAGE electrophoresis
• 2D Blue-Native/SDS PAGE electrophoresis
• SDS-PAGE electrophoresis
• In-gel activity assays
• RT-PCR
• Confocal life cell imaging
• Creation of transmitochondrial cybrids
• Western blotting and immunodetection
• Sucrose-gradient ultra-centrifugation and Mass spectrometry
• GATEWAY cloning
• Sequencing and restriction analysis
• Transient/stable transfection methods
• Cell culture

We advice students to follow at least one of the Courses on Molecular Biology, Cell biology and/or Biochemistry.

(II) Structure and Function of mitochondrial complex I in health and disease. 
(Prof. Dr. Uli Brandt, Dept. Pediatrics UMCN; Tel: 024-3667098;)

Research focuses on the structure and function of complex I (see research topic I) and how this is linked to the mitochondrial metabolism. Studies are performed both in the yeast Y. lipolytica that we have established as a genetic model specifically for this purpose and human cell lines. The work is based on recently solved structure of mitochondrial complex I from Y. lipolytica, the largest membrane protein complex ever analysed by X-ray crystallography.

Research question include: What is the mechanism of proton pumping and how is it driven by the reduction of ubiquinone? What is the role of accessory subunits in modulating the catalytic activity of complex I? How do mutations found in patients with complex I deficiency specifically affect the the enzyme structurally and functionally? How is the production of reactive oxygen species by complex I controlled? What is the impact of complex I deficiency on other mitochondrial complexes?

Current projects:

• The access pathways for ubiquinone n complex I
• The structural basis of the A/D transition of complex I
• The role of accessory subunits in complex I funcion
• Impact of complex I dysfunction on the mitochondrial complexome

During a traineeship students are able to get experienced with a divers spectrum of techniques:

• 1D Blue-Native PAGE electrophoresis
• 2D Blue-Native/SDS PAGE electrophoresis
• LC/ESI-Mass Spectrometry
• Proteomics / Complexome Profiling
• SDS-PAGE electrophoresis / Western blotting
• In-gel activity assays
• Yeast genetics
• Isolation of Mitochondria
• High Resolution Respirometry
• Activity assays for complex I
• RT-PCR
• RNA interference
• Transient/stable transfection methods
• Cell culture

(III) Cell biological consequences and mitigation of mitochondrial dysfunction.
(Dr. Peter Willems and Dr. Werner Koopman, Dept. Membrane Biochemistry NCMLS; tel: 024-3614589; )

Research focuses on the (patho)physiology of metabolic disease and mitochondrial dysfunction with special reference to genetic disorders of the OXPHOS system. Emphasis lies on the combination of biochemistry, molecular biology and advanced life cell imaging to assess the molecular mechanisms underlying the cellular consequences of mitochondrial dysfunction. Of particular interest for the latter is the search for therapeutics that can improve dysfunction. Measurements are performed on cell lines and primary cultures of patient skin fibroblasts and skeletal muscle myotubes. Research topics focus on:

• Mitochondrial and cellular Ca²⁺ and ATP homeostasis
• Mitochondrial network complexity and mitochondrial dynamics
• The role of mitochondria in oxidant generation
• Regulation and consequences of the above during normo- and pathophysiology

During a traineeship students are able to get experienced with the following techniques:

• Cell culture
• Cell transfection using baculoviral vectors
• Fluorescent proteins
• In-cell bioluminescence monitoring using aequorin and luciferase
• Video-rate UV calcium imaging using confocal microscopy
• Multispectral videomicroscopy of living cells
• Fluorescence recovery after photobleaching analysis
• Image processing, quantitative analysis and data modelling
• 1D Blue-Native PAGE electrophoresis
• SDS-PAGE electrophoresis
• In-gel activity assays
• Western blotting and immunodetection

(IV) Proteins involved in mtDNA maintenance/gene expression and nucleoid dynamics.
(Prof. Dr. Hans Spelbrink, Dept. Paediatrics UMCN; Tel: 024-3615191; )

Mitochondrial DNA (MtDNA) is organized in foci, termed nucleoids, as small assemblies containing 2-10 mtDNA copies and various proteins that are poorly conserved in evolution. Despite a renewed interest in mtDNA due to its involvement in human disease and ageing, there are still many fundamental questions surrounding faithful copying (replication), repair, inheritance and gene expression of mtDNA in humans.

Our research focuses on these questions in part via the identification and characterization of novel proteins in nucleoid biology and/or mitochondrial gene expression as well as the detailed characterization of selected proteins whose basic functions have already been established . Ultimately, the aim is to not only understand the very basic molecular biology of for example mtDNA replication/repair and gene expression but to also understand these processes at the cell biological and organismal level. This will not only provide fundamental insight in some of the least understood processes in the cell, it will also provide a framework to understand and possibly treat mtDNA disease.

More specifically, in our research we mainly focus on:

• The detailed structure/function characterization of the mitochondrial DNA helicase Twinkle
• Identification/characterization of potential novel mtDNA maintenance and gene expression proteins using comparative proteomics and downstream cell & molecular biology assays
• Dynamics of mtDNA maintenance and gene expression in situ

During a traineeship students are able to get experienced with a divers spectrum of techniques:

• RNA interference
• Protein biochemistry
• SDS-PAGE electrophoresis
• RT-PCR
• (Confocal) fluorescent life cell imaging
• Immuno fluorescence & other in situ fluorescent labeling techniques
• Western blotting and immunodetection
• Ultra-centrifugation and other separation/isolation methods and Mass spectrometry
• Molecular cloning
• Sequencing and restriction analysis
• Transient/stable transfection methods
• Cell culture

We advice students to follow at least one of the Courses on Molecular Biology, Cell biology and/or Biochemistry.

(V) Biochemical and genetic characterization of mitochondrial disease patients. 
(Dr. Richard Rodenburg, dept Pediatrics UMCN, tel 024-3614818, )

Mitochondria contain at least 1200 different proteins, and a large subset of these is directly or indirectly involved in mitochondrial energy metabolism. In approximately 10% of the corresponding genes, genetic defects leading to a mitochondrial disease have been identified to date. The discovery of new genetic defects is an ongoing effort in our lab. This research line focuses on the biochemical characterization of novel genetic defect that have been identified in mitochondrial disease patients using state of the art molecular genetic techniques. The primary goal is to establish the pathogenicity of new genetic defects. In addition, we aim to understand the mechanism by which mutations disturb mitochondrial function. The results of our research are not only important for understanding the ethiopathogenesis of mitochondrial disease, but also broadens our general understanding of the biochemical pathways involved in mitochondrial energy metabolism. Genetic defects  are complemented by lentiviral transduction, which involves gene cloning and heterologous expression in patient-derived fibroblasts and myoblasts, followed by verification of protein expression (Western blot analysis, microscopical analysis to check for subcellular protein localization) and functional analysis of mitochondrial energy metabolism using techniques such as ATP production rate analysis, substrate oxidation rate measurements, oxygen consumption rate measurements and enzyme activity measurements.

Cytochrome c oxidase (COX) represents the terminal and most highly regulated enzyme complex of the mitochondrial respiratory chain. As such, COX is regulated by the expression of COX subunits in isoforms and binding of allosteric effectors. Our research focuses on the identification of COX subunit isoforms and interacting proteins and of regulatory ligands, such as hormones, second messengers, nucleotides, and cations, and their impact on mitochondria and cell function and cell survival under hypoxic/ischemic and degenerative conditions in the central nervous system.

To evaluate the involvement of COX in sex-, brain region-, and cell type-specificity of certain neurodegenerative diseases, such as Parkinson's and Alzheimer's disease, and of ischemic stroke, studies are performed on different primary cell types isolated from different regions of the brain of different sexes of wild-type in comparison with transgenic and hypoxic/ischemic mouse models. We aim to identify differences of structural, functional, and regulatory characteristics of COX and their different impact on mitochondrial function and neural cell vulnerability in these different model systems.

• Identifying cytochrome c oxidase (COX) isoforms and interacting proteins under patho- vs. physiological conditions
• Evaluation of the role of COX isoform expression for mitochondrial signaling (ATP, ROS) and neural cell function and survival under hypoxic and degenerative conditions
• Effects of isoform- and interacting protein-specific knockdown on mitochondrial function and neural cell survival
• COX regulation defects in mtDNA patient cells
• Studying the effect of allosteric factors, such as ATP/ADP and hormones, on COX and mitochondria function and cell survival

Our aim is to understand if structural, functional, and regulatory features of COX play a causative or consequent role for the development of neurodegenerative diseases and ischemic stroke. By understanding the underlying molecular mechanisms we will not only provide an insight into fundamental mitochondrial and enzyme biochemistry but also identify key targets for therapeutic or protective intervention against mitochondrial and neurodegenerative diseases.

During a traineeship students are able to get experienced with a diverse spectrum of techniques:

• Primary brain cell culture: sex-, brain region-, and cell type-specific
• Transient and stable cell transfection
• siRNA knockdown techniques
• BNE and mass spectrometry
• Fluorescent detector protein expression & Fluorescence life cell imaging
• Immunohisto- and immunocytochemistry
• Molecular biological techniques: subcloning, qRT-PCR
• Biochemical methods: SDS-PAGE and Western Blotting
• Measuring mitochondrial function: polarography, ATP-/ROS-assays

We advise students to follow at least one of the Courses on Molecular Biology, Cell biology and/or Biochemistry.