Novel Cdnf/manf Protein Family: Molecular Structure, Expression and Neurotrophic Activity

1. Review of the literature .............................................................................................1 1.1. Neurotrophic factors: The concept ........................................................................1 1.2. Neurotrophic factors for midbrain dopaminergic neurons ....................................3 1.2.1. Neurotrophins..............................................................................................3 1.2.2. GDNF family ligands .................................................................................4 1.2.3. Effects of GDNF on midbrain dopaminergic neurons ................................7 1.2.4. Other trophic factors for midbrain dopaminergic neurons ..........................8 1.3. Midbrain dopaminergic system .............................................................................9 1.4. Parkinson’s disease ............................................................................................13 1.4.1. Mechanisms of dopaminergic neurodegeneration in PD ..........................14 1.4.2. Modelling of PD........................................................................................16 1.4.3. 6-OHDA model of PD...............................................................................17 1.4.4. GDNF is protective and restorative in PD models ....................................18 1.4.5. Neurotrophic factors as therapeutic proteins for PD .................................19 1.5. Mesencephalic astrocyte-derived neurotrophic factor (MANF) .........................20 1.5.1. MANF is a survival factor for midbrain dopaminergic neurons ...............20 1.5.2. MANF is induced by ER stress and protects against ischemic cell death 21 1.6. Saposin-like proteins (SAPLIPs) ........................................................................23 1.6.1. SAPLIPs have diverse functions ...............................................................23 1.6.2. Sphingolipid activator proteins (saposins) ................................................24 1.6.3. NK-lysin and granulysin ...........................................................................25 1.6.4. Membrane interactions of SAPLIPs .........................................................26 2. Aims of the study ......................................................................................................28 3. Materials and methods ............................................................................................29 3.1. DNA and RNA methods......................................................................................29 3.1.1. RNA isolation and reverse transcription ...................................................29 3.1.2. PCR, cloning and DNA sequencing ..........................................................29 3.1.3. Northern and in situ hybridization ............................................................31 3.2. Recombinant protein production .........................................................................31 3.2.1. CDNF production, purifi cation and SeMet labelling ................................31 3.2.2. MANF production and purifi cation...........................................................32 3.2.3. N-terminal sequencing and mass spectrometry ........................................33 3.2.4. Crystallization and structure determination ..............................................33 3.3. Immunological methods ......................................................................................33 3.3.1. Antibody production .................................................................................33 3.3.2. Tissue protein extracts and western blotting .............................................33 3.3.3. Immunohistochemistry..............................................................................33 3.4. Cell culture methods ...........................................................................................33 3.4.1. Primary neuronal cultures .........................................................................33 3.4.2. Transfection ...............................................................................................34 3.5. Animal models ....................................................................................................34 3.5.1. 6-OHDA rat model of Parkinson’s disease ...............................................34 3.5.2. Rat model of global forebrain ischemia or status epilepticus ...................34 4. Results ...................................................................................................................35 4.1. CDNF is a vertebrate paralogue of MANF (I) ....................................................35 4.2. CDNF and MANF are secreted proteins (I, II) ...................................................36 4.3. Protein production and purifi cation (I, II, III) .....................................................36 4.4. Crystal structure of CDNF and MANF (III) .......................................................37 4.5. CDNF expression in mammalian CNS (I) ..........................................................37 4.6. MANF expression in mammalian CNS (II) ........................................................39 4.7. MANF expression after epileptic and ischemic insults (II) ................................39 4.8. CDNF and MANF expression in non-neuronal tissues (I, II) .............................40 4.9. Neuroprotective effect of CDNF in 6-OHDA rat model of PD (I) .....................40 4.10. Neurorestorative effect of CDNF in 6-OHDA rat model of PD (I) ..................41 4.11. Studies on the survival promoting activity of CDNF on primary neuronal cultures (I) .........................................................................41 5. Discussion..................................................................................................................42 5.1. A possible dual role of CDNF and MANF..........................................................42 5.1.1. Secretion and ER localization of CDNF and MANF ................................42 5.1.2. CDNF and MANF: Lipid binding proteins? .............................................43 5.1.3. The putative membrane interaction of CDNF and MANF: Dimerization, conformational changes and pH effect? .............................43 5.1.4. The C-terminal domain: Cytoprotection against ER stress? .....................46 5.1.5. CDNF as potent as GDNF in 6-OHDA model of PD ...............................47 5.2. Novel neurotrophic factors for midbrain dopaminergic neurons ........................49 5.3. Roles of CDNF and MANF in non-neuronal tissues? ........................................50 5.4. CDNF and MANF as therapeutic proteins ..........................................................51 5.5. Receptors for CDNF and MANF are unknown ..................................................52 5.6. Evolutionarily conserved function? ....................................................................52 6. Conclusions ...............................................................................................................54 7. Acknowledgements ...................................................................................................55 8. References .................................................................................................................56 SELECTED ABBREVIATIONS