Death pathways activated in the neurotrophic factror-deprived neurons

1. REVIEW OF THE LITERATURE ..........................................................................1 1.1. Programed cell death ............................................................................................1 1.1.1. Programed cell death in the development of nervous system.....................1 1.2. Neurotrophic factors .............................................................................................2 1.2.1. Neurotrophin family ...................................................................................3 1.2.1.1. Nerve growth factor ......................................................................4 1.2.1.2. Brain-derived neurotrophic factor .................................................4 1.2.1.3. Neurotrophin-3 ..............................................................................5 1.2.2. Glial cell line-derived neurotrophic factor family .....................................5 1.3. GDNF as a neuroprotective trophic factor for midbrain dopaminergic neurons ..6 1.4. Classifi cation of cell death: apoptosis and necrosis..............................................8 1.5. Apoptotic pathways ............................................................................................10 1.5.1. Bcl-2 family ..............................................................................................10 1.5.2. Caspase family .........................................................................................13 1.5.3. Intrinsic or mitochondrial apoptotic pathway .........................................17 1.5.4. Extrinsic or death receptor apoptotic pathway ........................................18 1.5.4.1. Death receptors............................................................................18 1.5.4.2. Death receptor apoptotic pathway ...............................................19 1.6. Non-apoptotic programed cell death pathways ..................................................21 1.6.1. Autophagic cell death ...............................................................................21 1.6.2. Other non-apoptotic cell death pathways ................................................22 1.7. Model of neurotrophic factor-deprived neurons .................................................23 1.8. Active modes of death ........................................................................................24 1.8.1. Dependence receptors ..............................................................................25 1.8.2. Pro-neurotrophins and P75NTR ..................................................................25 1.9. One cell, multiple cell death pathways ...............................................................26 2. AIMS OF THE STUDY ...........................................................................................28 3. MATERIALS AND METHODS .............................................................................29 3.1. Cell cultures and survival assays (I, II, III and IV) ............................................29 3.2. Reverse transcription-polymerase chain reaction (RT-PCR) (IV) ......................29 3.3. Microinjection and transfections (I, II, III and IV).............................................30 3.4. Immunochemistry (I, IIIand IV) .........................................................................30 3.5. Immunoblot and co-immunoprecipitation (II, III and IV) ..................................31 3.6. Electron microscopy analysis (I) ........................................................................31 3.7. Statistical analysis (I, II, III and IV) ...................................................................31 4. RESULTS ..................................................................................................................32 4.1. GDNF-dependent sympathetic neurons die via a non-mitochondrial pathway which requires caspase activation (I) ...................................................32 4.2. The dependence receptor activity of TrkC is necessary for the death of NT-3-deprived sensory neurons (II) ...............................................33 4.3. A survival assay of transiently transfected dopaminergic neurons to analyze the apoptotic proteins in these neurons (III) .....................................34 4.4. GDNFor BDNF-deprived dopaminergic neurons die via a nonmitochondrial pathway which requires activation of death receptors and caspases (IV) ......................................................................................................35 5. DISCUSSION ...........................................................................................................38 6. CONCLUSIONS ......................................................................................................43 7. ACKNOWLEDGMENTS .......................................................................................44 8. REFERENCES .........................................................................................................45