Charakterisierungsstudien der biologischen und neurotrophen Eigenschaften des cerebral dopamine neurotrophic factor (CDNF)

Neurotophic factors enable the survival of neurons under pathological conditions and mediate novel proliferation of damaged neuronal cell structures. Neurotrophic factors are defined as secretory proteins. They are clustered into families according to structural similarities, receptors and signal transduction pathways. These neurotrophic protein families contain proteins of diverse functions. The neurotrophic abilities of CDNF - a member of the CDNF/MANF family - first have been discovered in vivo. Protection and proliferation of neurons were observed after intrastriatal application of CDNF in a rat model of neuronal degeneration. This protein family could therefore lead to a new concept in the therapy of neurodegenerative diseases. CDNF and MANF have a molecular weight of around 21 kDa. The amino terminus is formed by a globular saposin-like protein (SAPLIP) domain, whereas the carboxy terminus is related to scaffold attachment factors (SAF) and also contains a redox-active cysteine bridge presented in a CXXC motif. The biological function, receptors and molecular mechanisms involved in the neurotrophic effect are hence unknown. This work presents a set of experiments to characterize the properties of CDNF and its SAPLIP domain. The extensive biotechnological work of the performed experiments was conducted at CSL Behring, Marburg. The first part of this thesis investigates a proposed theory that the SAPLIP domain of CDNF could initiate a transduction effect. Due to their size and natural structure macromolecules are not able to pass cell membranes passively. Therefore only a few therapeutical substances have easy access to the interior of cells. In the 1980’s a special class of transporter peptides was discovered that can pass the cell membrane together with attached macromolecules. The Nterminal domain of CDNF does not contain the typical structure of these cell membranepenetrating peptides but it is related to saposins. Saposins and saposin-like domains of other proteins can bind lipids (e.g. for degradation in lysosomes) or even permeabilize cell membranes. These abilities lead to the proposal of the potential transduction effect of the SAPLIP domain of CDNF. CDNF fusion proteins were designed and produced biotechnologically to investigate the transduction effect of the SAPLIP domain. However, no transduction effect or association of CDNF with cell membranes could be detected. The second part shows the cellular localization of native CDNF which has been described as a secretory neurotrophic factor. Interestingly, the C-terminal KTEL sequence of CDNF is similar to the general ER retention sequence KDEL. We could show for the first time with different experimental methods that CDNF is retarded in the ER. Different CDNF mutants were designed to proof the importance of the KTEL retention signal. The CDNF mutant with the general ER retention sequence KDEL and native CDNF could not be detected in the supernatant of confluent cell cultures. In contrast, CDNF mutants with deleted, masked or otherwise altered C-terminus could be discovered in the medium as secreted proteins. This shows the importance of a free C-terminal KTEL sequence to investigate native CDNF localization and trafficking. The third part of the thesis was designed to endeavour the neurotrophic mechanisms of CDNF in vitro. Putative neuroprotective effects should be further investigated by molecular and biochemical methods. Different neuronal cell cultures were damaged by 4-hydroxydopamin or glutamate. Recombinant CDNF or the isolated SAPLIP domain were externally administered to investigate the protective effect of these proteins. In contrast to the formerly discovered protective effect in vivo no protective or proliferative effects of CDNF were observed in vitro.