Charakterisierungsstudien der biologischen und neurotrophen Eigenschaften des cerebral dopamine neurotrophic factor (CDNF)
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Philipps-Universität Marburg
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Abstract
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.
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Dates
Created: 2011Issued: 2012-08-29Updated: 2012-08-29
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Is part of: eb/2024/0241
Faculty
Fachbereich Pharmazie
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
LUHMES-ZellenCerebral dopamine neurotrphic factorHT-22 cellsHT-22 Zellen6-hydroxydopamineLUHMES cellsMesc cellsER-Retention6-HydroxydopaminProduction of recombinant proteinsHerstellung rekombinanter ProteineMesc-Zellen
DFG-subjects
Brain-derived neurotrophic factorCiliary neurotrophic factor
DDC-Numbers
610
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Barkholz, Michael: Charakterisierungsstudien der biologischen und neurotrophen Eigenschaften des cerebral dopamine neurotrophic factor (CDNF). : Philipps-Universität Marburg 2012-08-29. DOI: https://doi.org/10.17192/z2012.0792.
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This item has been published with the following license: In Copyright