Prions and autophagy [Elektronische Ressource] : effect of lithium on prion infection and role of basal autophagy in primary prion infection / Andreas R. Heiseke

Heiseke Andreas

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 23
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN
Lehrstuhl für Tierhygiene


Prions and autophagy: Effect of lithium on prion infection and
role of basal autophagy in primary prion infection

Andreas R. Heiseke

Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung
des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.


Vorsitzender: Univ.-Prof. Dr. H. H. D. Meyer

Prüfer der Dissertation:
1. Univ.-Prof. Dr. Dr. h.c. J. Bauer
2. Priv.-Doz. Dr. J. Beckers
3. Univ.-Prof. Dr. H. Schätzl


Die Dissertation wurde am 26.10.2009 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung
und Umwelt am 26.02.2010 angenommen. TABLE OF CONTENTS
TABLE OF CONTENTS
I. SUMMARY………………………………………………………………………………... 1
I.A ENGLISH VERSION……………………………………………………………………… 1
I.B GERMAN VERSION............................................................................................................ 3
II. INTRODUCTION…………………………………………………………………………. 5
II.A THE PRION PROTEIN……………………………………………………………………. 5
II.A.1 HISTORICAL BACKGROUND…………………………………………………………... 5
II.A.2 PRION DISEASES……………………………………………………………………… 6
II.A.2.1 HUMAN PRION DISEASES…………………………………………………………… 7
II.A.2.2 ANIMAL PRION DISEASE……………………………………………………………. 10
II.A.3 PRION GENE STRUCTURE……………………………………………………………... 12
II.A.4 FUNCTION OF THE PRION PROTEIN……………………………………………………. 12
C SC
II.A.5 STRUCTURAL AND BIOCHEMICAL CHARACTERISTICS OF PRP AND PRP …………… 14
II.A.6 CELL BIOLOGY AND LIFE-CYCLE OF PRP……………………………………………... 16
II.A.7 MECHANISMS OF PRION CONVERSION………………………………………………... 19
II.A.8 PRION STRAINS AND SPECIES BARRIER………………………………………………. 21
II.A.9 THERAPEUTIC APPROACHES…………………………………………………………. 23
II.B AUTOPHAGY…………………………………………………………………………… 25
II.B.1 TYPES OF AUTOPHAGY……………………………………………………………….. 25
II.B.2 AUTOPHAGIC SIGNAL TRANSDUCTION……………………………………………….. 26
II.B.2.1 MTOR-DEPENDENT AND -INDEPENDENT INDUCTION OF AUTOPHAGY……………… 26
II.B.2.2 AUTOPHAGOSOME FORMATION……………………………………………………. 29
II.B.3 ROLE OF AUTOPHAGY IN HEALTH AND DISEASE……………………………………… 30
II.B.3.1 AUTOPHAGY IN CELLULAR HOMEOSTASIS, STRESS INDUCED DIFFERENTIATION
AND DEVELOPMENT…………………………………………………………………………. 32
II.B.3.2 AUTOPHAGY IN CELL SURVIVAL AND CELL DEATH………………………………… 32
II.B.3.3 AUTOPHAGY AND CANCER…………………………………………………………. 33
II.B.3.4 AUTOPHAGY IN INFECTION AND IMMUNITY………………………………………… 34
II.B.3.5 AUTOPHAGY AND NEURODEGENERATION………………………………………….. 35
II.B.4 AUTOPHAGY AND PRIONS……………………………………………………………. 36
II.C OBJECTIVE……………………………………………………………………………... 38
III. MATERIALS AND METHODS…………………………………………………………… 39
III.A. MATERIALS………………………………………………………………………….. 39
TABLE OF CONTENTS
III.A.1 CHEMICALS………………………………………………………………………… 39
III.A.2 BUFFERS AND SOLUTIONS…………………………………………………………… 40
III.A.3 ANTIBIOTICS AND BACTERIA……………………………………………………….. 40
III.A.4 ENZYMES…………………………………………………………………………… 41
III.A.5 INHIBITORS…………………………………………………………………………. 41
III.A.6 ANTIBODIES………………………………………………………………………… 41
III.A.7 OLIGODESOXYNUCLEOTIDES……………………………………………………….. 43
III.A.8 PLASMIDS…………………………………………………………………………… 43
III.A.9 EUCARYOTIC CELL LINES…………………………………………………………… 44
III.A.10 CELL CULTURE MEDIA AND SUPPLEMENTS………………………………………… 45
III.A.11 KITS……………………………………………………………………………….. 45
III.A.12 INSTRUMENTS AND ACCESSORIES………………………………………………… 45
III.A.13 COMPUTING PROGRAMS…………………………………………………………… 47
III.B METHODS…………………………………………………………………………….. 47
III.B.1 BIOLOGICAL SAFETY……………………………………………………………….. 47
III.B.2 MOLECULAR BIOLOGICAL METHODS……………………………………………….. 47
III.B.2.1 QUANTIFICATION OF NUCLEIC ACID………………………………………………. 47
III.B.2.2 AMPLIFICATION OF PLASMID DNA……………………………………………….. 48
III.B.2.2.1 PREPARATION OF CHEMICALLY COMPETENT BACTERIA………………………… 48
III.B.2.2.2 HEAT SHOCK TRANSFORMATION OF E. COLI WITH PLASMID DNA……………… 48
III.B.2.2.3 ISOLATION OF PLASMID DNA…………………………………………………… 49
III.B.2.3 ISOLATION OF TOTAL CELLULAR RNA……………………………………………. 49
III.B.2.4 REAL-TIME (RT)-PCR……………………………………………………………. 50
III.B.3 PROTEIN BIOCHEMICAL METHODS…………………………………………………... 50
III.B.3.1 PREPARATION OF POSTNUCLEAR LYSATES………………………………………... 50
III.B.3.2 PROTEINASE K (PK)-DIGESTION………………………………………………….. 51
III.B.3.3 DETERGENT SOLUBILITY ASSAY…………………………………………………... 52
III.B.3.4 SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS
(SDS-PAGE)………………………………………………………………………………. 52
III.B.3.5 WESTERN BLOT (IMMUNOBLOT)………………………………………………….. 53
III.B.3.6 BAND INTENSITY QUANTIFICATION BY IMAGEQUANT TL………………………… 54
III.B.3.7 STATISTICAL ANALYSIS…………………………………………………………… 54
III.B.4 CELL BIOLOGICAL METHODS………………………………………………………... 55
III.B.4.1 THAWING OF CELLS……………………………………………………………….. 55
TABLE OF CONTENTS
III.B.4.2 CULTIVATION AND PASSAGING OF CELLS…………………………………………. 55
III.B.4.3 CRYOCONSERVATION OF CELLS…………………………………………………… 55
III.B.4.4 DETERMINATION OF CELL NUMBERS……………………………………………… 55
III.B.4.5 TREATMENT OF CELLS WITH CHEMICAL COMPOUNDS…………………………….. 56
III.B.4.6 TRANSIENT TRANSFECTION OF CELLS……………………………………………... 56
III.B.4.7 TRANSDUCTION OF CELLS………………………………………………………… 57
III.B.4.7.1 PRODUCTION OF RECOMBINANT LENTIVIRUS……………………………………. 57
III.B.4.7.2 TRANSDUCTION OF CELLS WITH RECOMBINANT LENTIVIRUS……………………. 57
III.B.4.8 PRION INFECTION OF CELL LINES………………………………………………….. 57
III.B.4.8.1 PREPARATION OF BRAIN HOMOGENATE…………………………………………. 57
III.B.4.8.2 INOCULATION OF CELLS WITH BRAIN HOMOGENATE……………………………. 57
III.B.4.9 TRYPAN BLUE ASSAY……………………………………………………………… 58
III.B.4.10 CONFOCAL LASER MICROSCOPY ANALYSIS (IMMUNOFLUORESCENCE)…………... 58
III.B.4.11 FLUORESCENCE-ACTIVATED CELL SORTING (FACS)……………………………. 59
III.C ANIMAL EXPERIMENTS………………………………………………………………... 60
IV. RESULTS………………………………………………………………………………. 61
SC
IV.A AUTOPHAGY INDUCTION MEDIATES REDUCTION OF PRP ……………………………. 61
IV.A.1 LITHIUM INDUCES AUTOPHAGY IN PRION-INFECTED CELLS…………………………. 61
SC
IV.A.2 LITHIUM ENHANCES CLEARANCE OF PRP IN NEURONAL AND
NON-NEURONAL CULTURED CELLS………………………………………………………….. 64
SCIV.A.3 LITHIUM-INDUCED AUTOPHAGY MEDIATES REDUCTION OF PRP ………………….. 67
SC
IV.A.4 AUTOPHAGY-INDUCING COMPOUNDS REDUCE PRP ……………………………….. 69
SC
IV.A.5 SEVERAL LITHIUM SALTS REDUCE PRP AND INDUCE AUTOPHAGY………………… 69
CIV.A.6 LITHIUM REDUCES THE LEVEL OF PRP ……………………………………………... 72
IV.A.7 THERAPEUTIC POTENTIAL OF AUTOPHAGY INDUCTION IN PRION DISEASE
AS MONITORED IN BIOASSAY………………………………………………………………... 74
IV.B ROLE OF BASAL AUTOPHAGY IN PRIMARY PRION INFECTION………………………….. 75
IV.B.1 AUTOPHAGOSOME FORMATION IS ACTIVATED UPON PRIMARY PRION INFECTION…… 75
IV.B.2 AUTOPHAGY-DEFICIENT MOUSE EMBRYONIC FIBROBLASTS (MEFS) ARE LESS
SUSCEPTIBLE TO PRIMARY PRION INFECTION COMPARED TO WILD-TYPE MEFS…………….. 80
IV.B.3 BASAL AUTOPHAGY ENHANCES PRIMARY PRION INFECTION………………………... 81
-/-
IV.B.3.1 REINTRODUCTION OF ATG5 IN MEFATG5 CELLS……………………………….. 81
IV.B.3.2 INCREASED SUSCEPTIBILITY TO PRIMARY PRION INFECTION UPON
-/-
REINTRODUCTION OF ATG5 IN MEFATG5 CELLS…………………………………………. 83
TABLE OF CONTENTS
V. DISCUSSION…………………………………………………………………………….. 86
SC
V.A. AUTOPHAGY INDUCTION MEDIATES REDUCTION OF PRP ……………………………. 86
SC
V.A.1. LITHIUM REDUCES PRP ……………………………………………………………. 86
V.A.2. INDUCTION OF AUTOPHAGY BY LITHIUM MEDIATES REDUCTION OF
SC
PRP IN CELLS PERSISTENTLY INFECTED WITH PRIONS…………………………………….. 87
C
V.A.3. LITHIUM REDUCES THE AMOUNT OF PRP …………………………………………… 89
SC
V.A.4. SEVERAL AUTOPHAGY INDUCERS REDUCE PRP IN CELLS
PERSISTENTLY INFECTED WITH PRIONS……………………………………………………… 90
V.A.5. mTOR-DEPENDENT VERSUS mTOR-INDEPENDENT AUTOPHAGY INDUCTION……….. 91
V.A.6. IS THERE THERAPEUTIC POTENTIAL OF AUTOPHAGY INDUCTION IN
TREATMENT OF PRION DISEASE? …………………………………………………………… 92
V.B. ROLE OF BASAL AUTOPHAGY IN PRIMARY PRION INFECTION…………………………... 94
V.B.1. MULTIPLE ROLES OF AUTOPHAGY IN PRIMARY PRION INFECTION? ………………….. 94
V.B.2. AUTOPHAGOSOME FORMATION IS ACTIVATED UPON PRIMARY PRION INFECTION……. 96
V.B.3. BASAL AUTOPHAGY PROMOTES PRIMARY PRION INFECTION………………………… 98
VI. ABBREVIATIONS……………………………………………………………………….. 101
VII. REFERENCE LIST……………………………………………………………………... 104
VIII. PUBLICATIONS……………………………………………………………………….. 136
IX. ACKNOWLEDGEMENT………………………………………………………………..... 137
X. CURRICULUM VITAE……………………………………………………………………. 138
SUMMARY
__________________________________________________________________________________________
I. SUMMARY
I.A ENGLISH VERSION
The formation of an abnormally folded, protease resistant isoform of the host-encoded
c
cellular prion protein (PrP ) is thought to be the responsible mechanism for prion diseases.
Sc cThis disease-associated agent, designated PrP , is derived from PrP by a post-translational
c
conformational change, and the presence of PrP is essential for development of prion disease.
c
The exact physiological function of PrP is