Quantification of isoprenoids in brain tissue [Elektronische Ressource] : cerebral regulation of FPP and GGPP in Alzheimer's Disease and aging / von Gero Peter Hooff

Gero Birke

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2010
• Nombre de lectures: 22
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Quantification of isoprenoids in brain tissue - Cerebral
regulation of FPP and GGPP in Alzheimer's Disease and
aging



Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften


vorgelegt beim Fachbereich Biochemie, Chemie und Pharmazie
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main









von
Gero Peter Hooff
aus Kempten





Frankfurt am Main (2010)
(D30)



vom Fachbereich 14 der


Johann Wolfgang Goethe – Universität als Dissertation angenommen.































Dekan: Prof. Dr. D. Steinhilber




Gutachter: Prof. Dr. W.E. Müller

Prof. Dr. M. Karas



Datum der Disputation: 14.04.2011
ABBREVIATIONS ...............................................................................................6
1. INTRODUCTION.............................................................................................9
1.1. Cholesterol ............................................................................................................................9
1.1.1. Biosynthesis ...................................................................................................................9
1.1.2. Cholesterol homeostasis in the brain............................................................................10
1.1.3. The role and function of brain cholesterol ...................................................................13
1.2. The physiology of the cell membrane .................................................................................13
1.3. Isoprenoids – Chemistry and Structure ...............................................................................15
1.3.1. Longer chain isoprenoids – dolichol and ubiquinone ..................................................16
1.3.2. Analytical approaches for the quantification of longer chain isoprenoids...................16
1.3.3. Farnesyl- and geranylgeranylpyrophosphate................................................................17
1.3.3.1. Role and function of FPP and GGPP ....................................................................17
1.3.3.2. Analytical approaches for the quantification of FPP and GGPP...........................18
1.4. Regulation of the mevalonate/isoprenoid/cholesterol pathway by sterols..........................20
1.5. Regulation of the mevalonate/isoprenoid/cholesterol pathway by non-sterols...................21
1.6. Protein prenylation – small GTPases ..................................................................................22
1.6.1. Prenylation – a post-translational modification............................................................22
1.6.2. Prenylated proteins – the small GTPases .....................................................................26
1.6.3. Analytical approaches for the quantification of farnesylated proteins.........................27
1.7. The aging brain and pathological implications of the MVA-pathway................................28
1.7.1. Cholesterol and aging...................................................................................................28
1.7.2. Cholesterol-precursors and -metabolites in aging........................................................29
1.7.3. Isoprenoids in aging and neurodegeneration................................................................30
1.7.3.1. Isoprenoids and aging............................................................................................30
1.7.3.2. Prenylated proteins, synaptic plasticity and aging ................................................31
1.7.3.3. The MVA-pathway and neurodegeneration..........................................................31
1.8. Alzheimer’s Disease............................................................................................................32
1.8.1. Prevalence and characteristics......................................................................................32
1.8.2. Etiology ........................................................................................................................32
1.8.3. Diagnosis......................................................................................................................33
1.8.4. Involvement of the MVA-pathway products and intermediates in AD .......................34
1.8.4.1. Cholesterol, statins and AD...................................................................................34
1.8.4.2. Isoprenoids and neurodegeneration.......................................................................35
1.8.4.3. Isoprenoids in AD .................................................................................................36
1.8.4.4. Prenylated proteins in AD.....................................................................................38
1.8.5. Prenylated proteins, synaptic plasticity and AD ..........................................................39
1.8.6. Prenylated proteins, oxidative stress and AD...............................................................40
1.9. Aims of the thesis................................................................................................................41
2. MATERIALS ..................................................................................................44
2.1. Chemicals ............................................................................................................................44
2.2. Western Blot Antibodies .....................................................................................................47
2.3. Kits ......................................................................................................................................47
2.4. Laboratory equipment and materials...................................................................................47
2.5. Software ..............................................................................................................................51
2.6. Buffers and Solutions..........................................................................................................51
1 2.6.1. General buffers.............................................................................................................51
2.6.2. Buffers and solutions for the isoprenoid isolation and derivatization..........................52
2.6.3. Western Blot buffers and solutions ..............................................................................53
2.6.4. Cell culture media and inhibitors .................................................................................55
3. METHODS ......................................................................................................56
3.1. Pre-column derivatization ...................................................................................................56
3.1.1. Standard prenylation assay (standard F-G-Assay) .......................................................56
3.1.1.1. Incubation protocol I .............................................................................................56
3.1.1.2. Incubation protocol II............................................................................................57
3.1.2. Prenylation assay following FPP and GGPP extraction...............................................57
3.2. Internal standard..................................................................................................................57
3.2.1. Synthesis.......................................................................................................................57
3.2.2. Product confirmation using thin layer chromatography...............................................58
1
3.2.3. H-NMR product confirmation ....................................................................................58
3.2.4. Preparative HPLC clean-up..........................................................................................58
3.3. FPP and GGPP extraction procedure for matrix samples ...................................................58
3.3.1. Sample preparation.......................................................................................................58
®3.3.2. Matrix assisted liquid/liquid extraction via Extrelut ..................................................59
®3.3.2.1. Generic Extrelut protocol ....................................................................................59
®3.3.2.2. Adapted Extrelut protocol ...................................................................................59
3.3.3. Solid phase extraction (SPE)........................................................................................59
3.3.3.1. Generic SPE protocol ............................................................................................59
3.3.3.2. Developed HLB protocol ......................................................................................60
3.3.4. Extraction procedure for in vitro cell culture experiments ..........................................60
3.4. An