Specialty course

animal-free methods in pharmacology and toxicology (6 weeks, full-time)

Coordination: Prof. Dr. M. Leist

Introduction:

We use cell cultures to examine mechanisms of damage and toxicity in the nervous system. For models of developmental neurotoxicity, we use stem cells that differentiate to neural cells (neural stem cells, neurons, glial cells). One basic part of the research projects is to characterize the developmental changes and to identify underlying mechanisms. New methods for better control and characterization of the differentiation are being developed. Thus, the course program will involve stem cell biology, neurodevelopment, epigenetic changes of developing cells and of cells exposed to stress and characterizations of toxicity and cell death. In addition we also have an interest in chemical biological processes related to post translational modifications of alpha synuclein and of cellular metabolism of MPP+, two molecules that can trigger Parkinson’s disease in experimental animals and man.

Our current research interest covers the following lines:

  1. Posttranslational oxidative modifications of alpha-synuclein: Synthesis of protein variants, characterization of their biological behavior with relation to effects on Parkinson’s disease.
  2. Development of a human neuronal model reflecting degeneration of dopaminergic neurons (relevant for Parkinson’s disease). Introduction of disease genes into human neurons by switchable vectors and examination of downstream pathomechanisms.
  3. Cocultures of glial cells and neurons to better study toxic processes e.g. of pesticides.
  4. Examination of the pathways that determine the generation of glial cells in the brain. Modulation of gliogenesis by toxicants and biological factors.
  5. Differentiation of embryonic stem cells to neural precursor cells. Characterization of epigenetic changes during this process, such as the modification of histones on certain promoters, and local alterations in chromatin density. Use of chromatin immunoprecipitation (ChIP) for characterization of chromatin changes.
  6. Differentiation of embryonic stem cells to neuronal precursor cells and mature neurons: examination of transcriptional changes associated with differentiation; examination of differentiation processes sensitive to certain toxicants.
  7. Examination of functional properties of “young neurons”, such as the migration of neural crest cells or the neurite growth of neural precursors as targets of toxicity.

All projects use cell culture technology, fluorescent staining and imaging technologies (high content automated imaging, pattern recognition and quantitative microscopy), FACS analysis, RT-PCR and ELISA methods. Some involve lentiviral vector construction, Western blotting, chromatin precipitations,…

 Course theory:

Our research unit was set up with the aim to introduce the 3R (reduce, replace, refine) principle of moderate and rational animal protection into the academic curriculum. Furthermore, the goal is to develop and judge alternative methods to animal experiments in the domain of consumer- and health protection with the help of interdisciplinary research. Alternative methods in the area of pharmacology and toxicology fall into three major domains: in silico methods (computer modeling based on quantitative structure-activity relationships), shift to human data (from epidemiological and clinical studies), and in vitro methods (use of cell and tissue cultures). Our major focus is on the latter domain. We are particularly interested in models for the pharmacology of neurodegenerative diseases, and models that would predict chronic and developmental neurotoxicity in humans.

The course will give a good theoretical basis on implications and applications of the 3R principle. It covers, amongst others, basic toxicological tests for chemicals, biologics, biotech products, drugs and nanoparticles. In addition, it introduces into the drug discovery processes and selected aspects of CNS pharmacology. Basic physiological and cell biological principles, and the biology of stem cells and stem cell differentiation are at the basis of many assays performed. As most topics are interdisciplinary, a good background in cell biology, molecular biology, immunology and basic human biology is required. Knowledge in pharmacology and toxicology is advisable, as alternative methods cut across these disciplines in many examples. The lectures will cover the whole range of 3R methods, but they focus particularly on specific scientific interests of our group. The lectures will be in English, if foreigners are present. In week 4 and 5 of the course, selected topics will be presented by students. This literature seminar will be based on English presentations. The discussion will clarify methodic aspects, and the relation of the chosen topic to actual research problems. Students are guided to critically judge third party data. Ethical aspects and the presentation style will also be addressed.

Literature: all material will be provided during the lectures. Some introductory literature on alternative methods and on the use of embryonic stem cells can be downloaded from our website.

Requirements:

Human Biology, Pharmacology and Toxicology I, and Biochemistry II, or equivalents to these lectures must have been followed and passed. Immunology would be desirable. Knowledge may be tested at he beginning of the course.

Course projects:

Projects are within the areas of CNS cell degeneration and re-generation, including pathobiochemical mechanisms implicated in chronic neurodegenerative disease and neurotoxicity. They will follow the research projects run at that time in the research group (see above). Knowledge of mechanisms will be translated into the development of new and improved in vitro test methods and potential treatment strategies of disease. The results of the student projects will be presented and discussed in the final week of the course.

Techniques involve advanced cell culture techniques, fluorescence microscopy, imaging algorithms, biochemical assays (Western, PCR, ELISA), plasmid preparation/analysis, transfection of cells, and isolation of CNS cell types. In addition, the use of relevant data handling and presentation software will be learned.

Exam:

To pass the course (for biological science students: obtain a “Schein”; for life science students: obtain a mark) the following requirements need to be met:

Active participation in the lectures, research project and presentation of the project results, power point presentation at the literature seminar, submission of a comprehensive work protocol in English language, delivered not later than four weeks after the end of the course.

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