3rd Biotechnology Symposium 2004 - Universität Leipzig

7.12 Suppression of apoptosis - A study of neuronal
cell death in three-dimensional retinal spheres
Antje Golbs, Andrée Rothermel, Winnie Weigel,
Randy Kurz, Markus Rüffer, Andrea A. Robitzki
Apoptosis plays a major role during embryonic development and in the formation of several
neurodegenerative diseases like Morbus Parkinson, Morbus Alzheimer, retinal degradations
leading to blindness such as macular degeneration and Retinitis Pigmentosa. In gene
silencing experiments with antisense caspase-3 cDNA transfection and overexpression of
the antiapoptotic protein Bcl-2, we tried to suppress programmed cell death in 3D retinalike
spheres, so called retinospheroids. First we determined the gene expression patterns
of caspase-3, Bcl-2 and Bcl-X L in retinae of the developing chicken embryo. Then, we correlated
our in vivo findings with the occurrence of apoptotic cells in rosetted spheroids. To
determine the rate of apoptosis cryosections of rosetted spheroids and in vivo retina, the
TUNEL-assay in combination with a DAPI cell nuclei staining was used. Additionally, we
investigated the temporal gene expression patterns of caspase-3, Bcl-2 and Bcl-X L in vivo
and in vitro by RT-PCR. The transfection experiments with antisense caspase-3 cDNA
antisense and sense Bcl-2 cDNA expression vectors were carried out using a calcium phosphate
transfection protocol. Both strategies showed the expected decrease in the number
of apoptotic cells in retinospheroids. Thus a first step to delay the progression of neuronal
degeneration and eye diseases is done.
Dr. Andrée Rothermel
Universität Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Professorship for Molecular Biological-Biochemical
Process Technology
and
Faculty of Biology, Pharmacy and Psychology
Institute of Biochemistry
E-Mail: andree.rothermel@bbz.uni-leipzig.de
www.uni-leipzig.de/~dmpt/
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