Yearbook 06-07


Ruth Schäfer
Home Institution Johann Wolfgang Goethe Universität, Frankfurt Host Institution University of California Los Angeles, David Geffen School of Medicine, Department of Neurology Mentor: Joanna C. Jen, M.D. Ph.D E-Mail: ruth@laborschaefer.de
Research Topic see Abstract
Personal Reactions to the U.S. Experience Even though the year is not over for me yet I can already say that spending a research year at UCLA was a great experience for me. The city of Los Angeles is enormous, there is so much you can see and do you never really spend a lazy minute. The weather in southern California is a dream for me being used to cold and rainy Frankfurt winters. The nature is overwhelming, the beach with the palm trees, the mountains with the sequoias and redwood trees, the dessert is really close and in the gardens you always find beautifully blooming flowers. LA offers a great cultural diversity. Museums, the opera, Ghetty Center, the philharmonic orchestra and also concerning the food since LA is a melting pot for many cultures living there and making life very interesting. Luckily I found a special place to live where I spend my time together with many other international students so I easily made good friends who shared many interests. At the University of California Los Angeles I worked in Professor Jen’s lab in the Department of Neurology. My PI turned out to be an enthusiastic scientist, a patient and very good teacher, a reliable supervisor and an understanding mentor for me. In the lab there we had a great working atmosphere. Young and interested researchers were always ready to help with any kinds of problems and became good friends.
Greatest Difficulties Encountered Luckily I did not have any major difficulties. Only some small ones like getting the DS-2019 form for my visa in time or trying to find an appropriate place to live with an affordable rent not too far from the University. What turns out to be very sad for me is that I still cannot get a clinical elective at UCLA because Uni Frankfurt does not have reciprocal relationships with UCLA. Even after providing a two-paged letter from our Deans Office in Frankfurt...
Most humorous incident Since I was living with many international students there were many situation related humorous incidents. Several of them had to do with some kind of language problems. A really funny moment that I will remember was when I went camping in November to Sequoia National Park with some of my friends. We drove for 5 hours and arrived in the dark. We knew there must be a campground so we drove up the mountains and found one. We went to sleep in our tents and it was already really cold… when I looked out of the tent the next morning I couldn’t believe my eyes: it was snowing in California! And I was sleeping in a tent and we were all freezing cold. Later that day we found out that we were camping 7000 feet high in the mountains and all around us were wildlife black bears…
Helpful Hints for Future Students Take the risk and spend a year doing research abroad. Get your visa forms as early as possible, don’t hesitate to contact your university and ask if they need any further materials. Get your Social Security Number that makes it easy to open a bank account and get a credit card… Buy your own car if you live in LA! Get a drivers license and be free. Travel as much as you can, there is a lot to see in California.
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Abstract on Research Topic
Mutations in the Gene of Human 3’-5’ DNA Exonuclease TREX1 Cause Retinal Vasculopathy with Cerebral Leukodystrophy

Institution:
University of California Los Angeles, David Geffen School of Medicine, Department of Neurology

Thanks to the help of: Hafsa Mamsa, Michael Kane, Lauren Sininger, Sonia Stoddart, Joanna Jen and Robert Baloh

Abstract:
Retinal vasculopathy with cerebral leukodystrophy (RVCL) patients share a prominent symptom: retinal vasculopathy characterized by teleangiectasias, microaneurysms and retinal capillary obliteration of the macula. In nine families heterozygous C-terminal frameshift mutations in the TREX1 gene were found. Mutant proteins retain exonuclease activity but lose the characteristic perinuclear localization of the wildtype. In the first described pedigree the illness was termed Cerebroretinal Vasculopathy (CRV), similar clinical features were found in two families and were named Hereditary Vascular Retinopathy (HVR) and another noted as Hereditary Endotheliopathy with Retinopathy, Nephropathy and Stroke (HERNS). The retinopathy often predated the neurological features, and in CRV and HERNS may include migraine headaches, multiple cerebral infarctions, white matter changes, cerebral pseudotumors and dementia. There is evidence of systemic vascular pathology such as micronodular cirrhosis and glomerular dysfunction. Ultrastructural studies of capillaries show a multi-lamellar subendothelial basement membrane. The RVCL mutation is located in the TREX1 gene. Despite clinical differences in the CRV and HVR pedigrees a heterozygous one base pair insertion leads to a V235 frameshift and premature stop. In HERNS a heterozygous four base pair insertion results in a frameshift at T249.
TREX1 is a DNA specific 3’ to 5’ exonuclease ubiquitously expressed in mammalian cells. It is thought to function as a homodimer with a preference for single-stranded DNA and mispaired 3’ termini. TREX1 is part of the SET complex which normally resides in the cytoplasm but translocates to the nucleus in response to oxidative DNA damage.
To investigate how the RVCL TREX1 mutants differ from WT we performed expression studies using confocal microscopy on cell cultures transfected with mutant TREX1 N-terminally epitope-tagged. The WT and the T249fs cDNA were PCR amplified from a HERNS patient and cloned into a mammalian expression vector pCMV-3TAG-6 with N-terminal FLAG (or myc for co-expression) epitope. The wildtype clone was mutagenized by QuickChange (Stratagene) to generate the V235fs and L287fs constructs. HeLa and RKO cells were transfected and fixed for immunostaining.
The WT TREX1 locates to the perinuclear region in an organized reticular pattern. In contrast the TREX 1 mutants were diffusely distributed in the cytoplasm and the nucleus.
These results suggest a perinuclear targeting signal within the carboxyl-terminus of TREX1. The lack of the C-terminus may prevent an interaction with the SET protein and therefore formation of the SET complex. The dissemination of TREX1 in the nucleus and cytoplasm may have enormous effects especially on endothelial cells. These families and the studies of their mutations should teach us about exonuclease function, homeostasis of the endothelium and events leading to premature vascular aging. Ongoing experiments such as the co-expression studies as well as stable transfected cell lines will help us understanding the molecular mechanisms leading to the severe phenotype.