Andreas Fischer

Home Institution
Humboldt-Universität Berlin

Host Institution
Department of Physiology and Biophysics, University of Colorado Health Sciences Center, Denver
Research Mentor: Margaret C. Neville, Ph.D.

E-Mail: andreas.fischer@student.hu-berlin.de

Research Topic
see Abstract
Personal Reactions to the U.S. Experience
I really enjoyed the chance to work in the lab of Margaret Neville, and I was very impressed by the warm and productive atmosphere there. Generally, although this was my first trip to the U.S. ever, I did not find things to be too different from Germany. Of course, there are certain peculiarities with respect to everyday life, but getting along with them is pretty easy. Denver turned out to be a beautiful place to stay, and living in a city that gets 300 days of sunshine a year definitely improves your mood. Furthermore, the landscape is really amazing and the skiing is considerably different from what I experienced before at the Fichtelberg.
Greatest Difficulties Encountered
Generally, my stay here was very well organized in advance by my mentor, and, as everybody else was very helpful, too, there were no real problems I had to deal with. However, bringing my transgenic mice from Germany to the U.S. was not that easy, and lots of administrative work had to be done. When they finally arrived in Denver, they seemed to be pretty jet lagged, too, and the females refused to get pregnant. Fortunately, in the end everything turned out fine.
Most humorous incident
During a conversation about pets, I mentioned that I had a bird once, on which I stepped inadvertently (no, this is not the funny part yet!), and when people asked me for its name, I truthfully told them that actually my mother chose its name to be Bubi. Although this is a pretty common name for pet birds in Germany, it is associated with something completely different in the U.S. At least now I know that I was obviously determined to end up in mammary gland biology.
Helpful Hints for Future Students
In case you are going to spend your time in Denver, feel free to contact me for any special information. Generally, make sure to establish a Spekulatius and Lebkuchen connection to Germany in time for the holiday season, as otherwise you might have a hard time finding these things in the U.S. The same goes for good chocolate. Opening a checking account at the Deutsche Bank might be a good idea, too. Withdrawing money at ATMs of the Bank of America is free that way, as long as you use your German EC card. But be careful, this doesn't work with a German credit card. Finally, avoid bringing lots of books with you from Germany, in order to prepare for examinations. You won't use them anyway and it just adds weight to your baggage.

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Abstract on Research Topic
Functional characterization of the RhoA effector molecule PKN in transgenic mice

Authors
Fischer(1,3), H. Stuckas(1), P. Schrade(2), S. Bachmann(2), M. Neville(3), Y. Ohashi(4), F. Theuring(1)
(1) Department of Pharmacology and Toxicology, Humboldt University, Berlin
(2) Department of Anatomy, Humboldt University, Berlin
(3) Department of Physiology and Biophysics, University of Colorado Health Sciences Center
(4) Schering Research Laboratories, Osaka

Institution
University of Colorado Health Sciences Center, Department of Physiology and Biophysics
Outline of the project
The small GTPase RhoA is implicated in a broad variety of cellular functions such as regulation of the actin cytoskeleton, cell cycle progression, transcriptional regulation and organization of intercellular contacts. Several lines of evidence suggest that this molecule plays a key role in metastasis and considerable efforts have been made to characterize the signaling pathways downstream of RhoA. Among its effector molecules, the ubiquitously expressed Ser/Thr kinase PKN has been implicated in transcriptional activation and cell cycle control, yet its physiological role remains unclear.
To elucidate the in vivo function of PKN, transgenic mice were generated that express a constitutively active mutant of PKN in their mammary glands under the control of the MMTV-LTR promoter. Two independent transgenic lines were established and females of both lines show a severe impairment of lactation as reflected by increased neonatal mortality and poor weight gain of pups suckled by transgenic mothers. Cross fostering experiments confirmed that this lactational failure is solely dependent on the genotype of the mother, but not of the pups, and an extensive histological analysis was carried out to characterize the morphology of the transgenic mammary glands.
Consistently, postpartum secretory alveoli in transgenic mice appeared smaller than in their wild type littermates and were full of a uniform eosinophilic substance, whereas no obvious alteration in the overall degree of proliferation could be observed in postpartum animals. As lactation progressed, there was a progressive loss of epithelial tissue in the mammary glands of transgenic mice consistent with precocious involution in these animals. TUNEL assays confirmed that this reduction in the epithelial compartment was paralleled by an increased number of apoptotic cells in transgenic animals. Electron microscopy was carried out to assess ultrastructural alterations in transgenic mammary epithelial cells. In addition to a severe disturbance in cellular organization, signs of retention of secretion products within the epithelial cells could be found. Consistent with this observation, it was demonstrated by immunohistochemistry that ß-casein, a major milk protein, accumulated abnormally in the transgenic epithelium. As PKN has been shown to interact with Phospholipase D, a key molecule for intracellular trafficking processes, additional experiments are planned to determine which of these trafficking processes might be affected in PKN transgenic animals.
Because RhoA has been reported to disrupt tight junctions in tissue culture systems, a second focus of this project is the assessment of tight junction function in PKN transgenic mice. Failure of tight junction closure in the murine mammary gland has been associated with lactational failure and apoptosis of mammary epithelial cells. By using up the teat injections of various tracer substances, the permeability of the mammary tight junctions will be analyzed and the intracellular distribution of the tight junction components ZO-1 and occludin will be assessed by immunohistochemistry.
Finally, as PKN is implicated in transcriptional activation, microarray experiments are planned to analyze changes in the transgenic animals on the transcriptional level. A substantial database of microarray results from various transgenic models, established in this lab provides the opportunity to compare transcriptional changes in PKN transgenic mice with other well-characterized models.
In conclusion, the analysis of the phenotype of PKN transgenic mice could lead to a better understanding of the RhoA signaling pathway in vivo, but also contribute to the current knowledge about mammary gland biology.