PROTEIN MISFOLDING AND DISEASE: PRINCIPLES AND PROTOCOLS
CONTENTS 1 STRUCTURAL AND CONFORMATIONAL PREREQUISITES OF AMYLOIDOGENESIS 1 ABSTRACT 2 INTRODUCTION 3 WHAT KIND OF DEFECTS IN THE SOLUBLE FOLDED STATE BOLSTER THE CONVERSION TO THE AMYLOIDOGENIC PHASE? 4 WHAT ARE THE CONFORMATIONAL PREREQUISITES FOR PARTIALLY FOLDED INTERMEDIATES TO BECOME THE AMYLOIDOGENIC SPECIES? 4 1 THE REQUIREMENT FOR PARTIAL UNFOLDING: FIBRILLOGENESIS OF GLOBULAR PROTEINS 4 1 1 TRANSTHYRETIN 4 1 2 ?2-MICROGLOBULIN 4 1 3 SERUM AMYLOID A PROTEIN 4 1 4 IMMUNOGLOBULIN LIGHT CHAINS 4 1 5 INSULIN 4 1 6 HUMAN LYSOZYME 4 1 7 ?-LACTALBUMIN 4 1 8 MONELLIN 4 2 THE REQUIREMENT FOR PARTIAL FOLDING: FIBRILLOGENESIS OF NATIVELY UNFOLDED PROTEINS 4 2 1 AMYLOID ?-PROTEIN 4 2 2 TAU PROTEIN 4 2 3 ?-SYNUCLEIN 4 2 4 AMYLIN 4 2 5 PROTHYMOSIN ? 4 3 CONFORMATIONAL PREREQUISITES FOR AMYLOIDOGENESIS: WHY A PREMOLTEN GLOBULE? 5 CONCLUDING REMARKS 6 ABBREVIATIONS REFERENCES CONTENTS 2 THE GENERIC NATURE OF PROTEIN FOLDING AND MISFOLDING 1 ABSTRACT 3 THE UNIVERSAL MECHANISM OF PROTEIN FOLDING 4 PROTEIN FOLDING AND MISFOLDING IN THE CELLULAR ENVIRONMENT 5 THE GENERIC NATURE OF AMYLOID FORMATION 6 COMMON FEATURES OF PROTEIN SELF-ASSEMBLY 7 GENERIC ASPECTS OF MISFOLDING DISEASES 8 COMMON STRATEGIES FOR THERAPEUTIC INTERVENTION 9 CONCLUDING REMARKS 10 ABBREVIATIONS ACKNOWLEDGMENTS 3 RELATIVE IMPORTANCE OF HYDROPHOBICITY, NET CHARGE, AND SECONDARY STRUCTURE PROPENSITIES IN PROTEIN AGGREGATION 1 ABSTRACT 3 IMPORTANCE OF HYDROPHOBICITY IN PROTEIN AGGREGATION 3 1 ANALYSIS OF FOUR REPRESENTATIVE CASES 3 1 1 A? 3 1 2 ?-SYN 3 1 3 PHF43 (FRAGMENT FROM ?) 3 1 4 ACP 3 2 PROTEIN AGGREGATION IS PROMOTED BY HYDROPHOBIC REGIONS OF A SEQUENCE 4 IMPORTANCE OF CHARGE IN PROTEIN AGGREGATION 4 1 REDUCTION OF THE NET CHARGE OF A PROTEIN INCREASES ITS PROPENSITY TO AGGREGATE 4 2 AGGREGATION IS FAVORED BY MACROMOLECULES WITH AN OPPOSITE CHARGE 4 3 CHARGE INTERACTIONS MODULATE, RATHER THAN PROMOTING SPECI? CALLY, AGGREGATION 5 IMPORTANCE OF THE PROPENSITY TO A FORM SECONDARY STRUCTURE IN PROTEIN AGGREGATION 5 1 SEQUENCES WITH A HIGH PROPENSITY TO FORM ?-STRUCTURES ARE HIGHLY AMYLOIDOGENIC 5 2 SEQUENCES WITH A HIGH PROPENSITY TO FORM ?-HELICAL STRUCTURES EXHIBIT POOR AMYLOIDOGENICITY 6 MUTATIONS MODULATE AGGREGATION AS A RESULT OF THEIR EFFECTS ON SIMPLE PHYSICOCHEMICAL DETERMINANTS 7 AMINO ACID SEQUENCES HAVE EVOLVED TO TAKE INTO ACCOUNT THE IN? UENCE OF HYDROPHOBICITY, CHARGE, AND ?-SHEET PROPENSITY IN PROTEIN AGGREGATION CONTENTS 8 OTHER FACTORS INVOLVED IN PROTEIN AGGREGATION 9 IS PROTEIN AGGREGATION DRIVEN BY SPECI? C SEQUENCES? 10 FUTURE PERSPECTIVES 11 ABBREVIATIONS 4 CYTOTOXIC INTERMEDIATES IN THE FIBRILLATION PATHWAY: AB OLIGOMERS IN ALZHEIMER’S DISEASE AS A CASE STUDY 1 ABSTRACT 2 AD IS A DEMENTIA INVOLVING THE FIBRILLOGENIC A? PEPTIDE 3 WHY THE FIBRIL-BASED CASCADE HYPOTHESIS UNRAVELED: A SINGULAR ILLUSTRATION WITH A TRANSGENIC MOUSE AD MODEL 4 IF NOT FIBRILS, WHAT? DISCOVERY OF A?’S HIDDEN TOXINS 5 OLIGOMERS HAVE PROFOUND NEUROLOGICAL IMPACT, ACCOUNTING FOR REVERSIBILITY OF MEMORY LOSS 6 HOW OLIGOMERS ATTACK NEURONS—A MOLECULAR MECHANISM FOR WHY AD IS SPECI? C FOR MEMORY LOSS 7 IMMEDIATE CONSEQUENCES OF OLIGOMER BINDING: SIGNAL TRANSDUCTION TARGETS 8 CASCADING CONSEQUENCES—CAN OLIGOMER-INDUCED SYNAPSE DYSFUNCTION LEAD TO SYNAPSE DESTRUCTION AND NEURON DEATH? 9 IN VIVO EXPERIMENTAL SUPPORT FOR SYNAPTOTOXIC OLIGOMERS: DATA FROM MOUSE MODELS OF EARLY AD 10 CLINICAL VALIDATION—OLIGOMERS IN HUMAN BRAIN, ELEVATED UP TO 70-FOLD IN AD 11 NEW “OLIGOMER-DRIVEN” AMYLOID HYPOTHESIS 12 MECHANISMS OF A? OLIGOMERIZATION AND FIBRILLOGENESIS 13 PATHOGENIC A? OLIGOMERS—FIRST OF MANY? ALL PROTEINS LIKELY HAVE THE CAPACITY TO OLIGOMERIZE 14 THERAPEUTICS AND DIAGNOSTICS—NEW STRATEGIES 15 ABBREVIATIONS POTENTIAL CON? ICTS 5 GLYCOSAMINOGLYCANS, PROTEOGLYCANS, AND CONFORMATIONAL DISORDERS 1 ABSTRACT 2 BIOCHEMICAL PROPERTIES OF PROTEOGLYCANS AND GLYCOSAMINOGLYCANS 3 NEURODEGENERATIVE DISEASES ARE PROTEIN CONFORMATIONAL DISORDERS 3 1 AD 3 2 PD 3 3 PRION DISEASES CONTENTS 4 PROTEOGLYCANS CONTRIBUTE TO PROTEIN MISFOLDING IN CONFORMATIONAL PROTEIN DISORDERS OUTSIDE THE NERVOUS SYSTEM 4 1 TYPE 2 DIABETES 4 2 IN? AMMATION-ASSOCIATED AA AMYLOIDOSIS 4 3 ?2-MICROGLOBULIN-RELATED AMYLOIDOSIS 5 CONCLUSIONS 6 APOLIPOPROTEINS IN DIFFERENT AMYLOIDOSES 1 ABSTRACT 3 MOLECULAR CHARACTERISTIC OF APOLIPOPROTEINS INVOLVED IN AMYLOIDOSES 3 1 APOLIPOPROTEIN A 3 2 APOLIPOPROTEIN E 3 3 APOLIPOPROTEIN J 4 ROLE OF APOLIPOPROTEINS IN PATHOLOGICAL MECHANISM OF AD AMYLOIDOSIS 4 1 EFFECT OF APO E ON A? FIBRILLIZATION AND DEPOSITION 4 2 ROLE OF APOLIPOPROTEINS IN A? TRAF? CKING ACROSS THE BBB, IN THE SERUM AND IN THE CSF 4 3 APOLIPOPROTEINS AND NEURONAL PATHOLOGY IN AD 4 4 APOLIPOPROTEINS AND CEREBRAL AMYLOID ANGIOPATHY 5 APOLIPOPROTEINS IN PRION DISEASES 6 APOLIPOPROTEINS IN OTHER AMYLOIDOSES 7 APOLIPOPROTEINS AS A SUBSTRATE OF AMYLOID FIBRILS 8 APOLIPOPROTEINS AS A THERAPEUTIC TARGET IN AMYLOIDOSES 9 ABBREVIATIONS 7 OXIDATIVE STRESS AND PROTEIN DEPOSITION DISEASES 1 ABSTRACT 2 INTRODUCTION TO OXIDATIVE AND NITRATIVE STRESSES 3 OXIDATIVE AND NITRATIVE STRESSES IN NEURODEGENERATIVE DISEASES WITH PROTEIN DEPOSITS 3 1 ALZHEIMER’S DISEASE 3 1 1 OXIDATIVE MODI? CATIONS OF TAU ARE ASSOCIATED WITH PROTEIN DEPOSITION AND NEURODEGENERATION 3 2 OXIDATIVE PROTEIN DEPOSITION IN TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES 3 3 OXIDATIVE STRESS AND SYNUCLEINOPATHIES 4 ABBREVIATIONS CONTENTS 8 CHAPERONE AND CONFORMATIONAL DISORDERS 8 1 CHAPERONE SUPPRESSION OF AGGREGATED PROTEIN TOXICITY 1 ABSTRACT 2 PROTEIN FOLDING AND MISFOLDING 3 CELLULAR QUALITY CONTROL 3 1 THE MOLECULAR CHAPERONES 3 2 THE UBIQUITIN PROTEASOME 4 MUTATIONS IN MOLECULAR CHAPERONES RESPONSIBLE FOR HUMAN DISEASE 5 PROTEIN MISFOLDING DISEASES 5 1 AD 5 2 PD 5 3 ALS 5 4 POLYGLUTAMINE EXPANSION DISEASES 6 PROTEIN AGGREGATES: A COMMON DENOMINATOR OF NEURODEGENERATIVE DISEASE 7 MOLECULAR CHAPERONES: KEY REGULATORS OF PROTEIN AGGREGATION AND TOXICITY 7 1 AD AND RELATED DEMENTIAS 7 2 PD 7 3 FALS 7 4 POLYGLUTAMINE EXPANSION DISEASES 8 CHAPERONES AS A POTENTIAL DRUG TARGET 8 1 CHEMICAL CHAPERONES 8 2 DRUGS THAT UPREGULATE CHAPERONE EXPRESSION 8 2 MECHANISMS OF ACTIVE SOLUBILIZATION OF STABLE PROTEIN AGGREGATES BY MOLECULAR CHAPERONES 1 ABSTRACT 2 CHOOSING BETWEEN NATIVE FOLDING AND MISFOLDING 3 MANY MOLECULAR CHAPERONES CAN PREVENT PROTEIN AGGREGATION 4 SOME ATPASE CHAPERONES CAN SOLUBILIZE AND REACTIVATE STABLE PROTEIN AGGREGATES 5 PREVENTION OF AGGREGATION IS NOT REQUIRED FOR CHAPERONE-DEPENDENT PROTEIN REFOLDING 6 ATPASE CHAPERONES CAN UNFOLD MISFOLDED PROTEINS 7 RING-SHAPED CHAPERONE OLIGOMERS CAN USE POWER STROKES TO ACTIVELY UNFOLD AGGREGATES 8 INDIVIDUAL CHAPERONE MONOMERS CAN USE RANDOM MOTIONS TO ACTIVELY UNFOLD AGGREGATES 9 CONCLUSION: THE SUCCESSIVE LINES OF DEFENSE AGAINST PROTEIN AGGREGATION AND DISEASES CONTENTS 10 ABBREVIATION 9 THE AGGRESOME: PROTEASOMES, INCLUSION BODIES, AND PROTEIN AGGREGATION 1 ABSTRACT 3 CHARACTERISTICS OF AGGRESOMES 3 1 AGGRESOMES ARE COMPOSED OF AGGREGATED, UNDEGRADED PROTEIN 3 2 AGGRESOMES CAN BE UB POSITIVE OR UB NEGATIVE 3 3 AGGRESOME FORMATION IS AN MT AND DYNEIN–DYNACTIN-DEPENDENT PROCESS 3 4 AGGRESOMES FORMATION AT THE CENTROSOME 3 5 AGGRESOMES ARE ASSOCIATED WITH REARRANGEMENTS IN IFS 4 EXAMPLES OF AGGRESOMES IN HUMAN HEALTH AND DISEASE 4 1 AGGRESOMES THAT FORM AS PART OF A NORMAL PROCESS 4 1 1 DALIS (DENDRITIC CELL AGGRESOME-LIKE INDUCED STRUCTURES) 4 1 2 GLUTAMATE RECEPTOR SUBUNIT 1 (GLUR1) AGGRESOMES 4 1 3 STIGMOID BODIES: 5-HT7 RECEPTORS AND AROMATASE 4 2 AGGRESOMES THAT FORM AS PART OF A DISEASE PROCESS 4 2 1 AGGRESOMES AND VIRAL FACTORIES 4 2 2 MALLORY BODIES: HEPATIC DISORDERS AND CYTOKERATIN 4 2 3 RETINITIS PIGMENTOSA: RHODOPSIN 4 2 4 DESMIN-RELATED MYOPATHY (DRM): ?,?-CRYSTALLIN 4 2 5 PRION DISORDERS: PRION PROTEIN 4 2 6 BUNINA BODIES: ALS AND SOD1 4 2 7 HUNTINGTON’S DISEASE: EXPANDED CAG REGION OF HUNTINGTON 4 2 8 CHARCOT-MARIE-TOOTH DISEASE: PERIPHERAL MYELIN PROTEIN 22 (PMP22) 4 2 9 CATARACTS: CONNEXIN 50, 43 4 2 10 PARKINSON’S DISEASE: LEWY BODIES/PARKIN/?-SYNUCLEIN 5 MECHANISMS OF AGGRESOME FORMATION 5 1 PROTEASOME BIOLOGY: SUBSTRATE COMPETITION 5 2 CELLULAR NEUROBIOLOGY: THE ROLE OF THE UB–PROTEASOME PATHWAY IN NEURONS 5 3 THE BIOPHYSICAL PROCESS OF FIBRIL FORMATION 6 FUTURE DIRECTIONS 7 ABBREVIATIONS 10 PROTEIN AGGREGATION, ION CHANNEL FORMATION, AND MEMBRANE DAMAGE 1 ABSTRACT CONTENTS 3 ALZHEIMER’S DISEASE (A?) 4 PRION (PRP) CHANNELS 5 TYPE II DIABETES MELLITUS AND ISLET AMYLOID POLYPEPTIDE (IAPP, AMYLIN) 6 ?-SYNUCLEIN AND PARKINSON’S DISEASE 7 POLYGLUTAMINE AND TRIPLET REPEAT DISEASES 8 MECHANISMS OF MEMBRANE-MEDIATED DAMAGE 8 1 PLASMA MEMBRANES 8 2 MITOCHONDRIAL MEMBRANES 8 3 OTHER INTRACELLULAR MEMBRANES 8 4 OTHER AMYLOID PEPTIDES 11 VISUALIZATION OF PROTEIN DEPOSITS IN VIVO 11 1 CONGO RED STAINING OF AMYLOID: IMPROVEMENTS AND PRACTICAL GUIDE FOR A MORE PRECISE DIAGNOSIS OF AMYLOID AND THE DIFFERENT AMYLOIDOSES 1 ABSTRACT 2 AMYLOIDOSIS 3 IDENTI? CATION OF AMYLOID USING DYES 3 1 INTRODUCTION 3 2 STAINING OF AMYLOID BEFORE 1922 3 3 CR AS A DIAGNOSTIC TOOL (SINCE 1922) 3 4 SOME CURRENT STAINING PROTOCOLS USING CR 3 5 STAINING OF AMYLOID-LIKE FIBRILS WITH CR 3 6 CR AS A FLUOROCHROME 3 7 THIO? AVIN 3 8 OTHER DYES AND CR ANALOGUES 4 THE CHEMICAL STRUCTURE OF CR AND SOME PROPERTIES 4 1 HISTORY AND CHEMISTRY 4 2 CHARACTERISTICS OF CR CRYSTALS 4 3 CONCERNING THE VALUE OF THE GREEN POLARIZATION COLOR 4 4 COLORED ANISOTROPY AFTER BINDING OF CR 4 5 MECHANISM OF CR BINDING TO AMYLOIDS 5 CONCERNING THE SPECI? CITY OF CR 6 CONCERNING THE PRACTICAL USE OF CR 6 1 THE QUALITY OF EQUIPMENT 6 2 THE QUALITY AND KIND OF THE BIOPSY 6 3 THE SIZE OF THE BIOPSY AND THE SAMPLING ERROR 6 4 THE QUALITY OF TISSUE SECTIONS AND MINUTE AMYLOID DEPOSITS 6 5 THE QUALITY OF STAINING 6 6 IMBIBITION OF SERUM AND TISSUE PROTEINS 6 7 RELATIVE INSENSITIVITY OF THE CONVENTIONAL CR STAINING PROCEDURE CONTENTS 6 8 INCREASED SENSITIVITY OF THE CR PROCEDURE BY IMMUNOHISTOCHEMISTRY (CRIC) 6 9 INCREASED SENSITIVITY USING CRF 6 10 CONCERNING THE RECIPROCAL PROPERTIES OF SENSITIVITY AND SPECI? CITY 6 11 THE POLARIZATION SHADOW 6 12 INCONCLUSIVENESS OF A NEGATIVE AMYLOID DIAGNOSIS 6 13 PRECISION OF THE DIAGNOSIS AND COURTESY TOWARD THE CLINICIAN 7 CHEMICAL IDENTI? CATION OF AMYLOIDOSIS 7 1 BEFORE THE CHEMICAL IDENTI? CATION 7 2 CHEMICAL CLASSI? CATION 7 3 AMYLOID TYPING IN CLINICOPATHOLOGIC PRACTICE 7 4 IMMUNOHISTOCHEMICAL CLASSI? CATION OF AMYLOIDS 7 5 MICROEXTRACTION FOLLOWED BY AN AMINO ACID SEQUENCE 8 ADVICE FOR THE IMMUNOHISTOCHEMICAL CLASSI? CATION OF AMYLOIDS 8 1 INTRODUCTION 8 2 APPROVED ANTIBODIES AND CONTROLS 8 3 SETS OF ANTIBODIES 8 4 PRESTAINING WITH CR 8 5 SERIAL SECTIONS 8 6 OTHER AMYLOID COMPONENTS 9 FROM BENCH TO BEDSIDE: AN ALGORITHM FOR A RELIABLE DIAGNOSIS 9 1 CLASSI? CATIONS BEFORE THE CHEMICAL NATURE OF AMYLOID WAS KNOWN 9 2 CLASSI? CATION ACCORDING TO THE CHEMICAL NATURE OF AMYLOID PROTEINS 9 3 AN IMPORTANT REMARK POINTING TO THE CORRECT HIERARCHY OF DIAGNOSING AMYLOIDOSIS 9 4 DIAGNOSING AMYLOIDOSIS CORRECTLY WITH RESPECT TO THERAPY 10 QUANTI? CATION OF AMYLOID 11 NOVEL TECHNIQES IN AMYLOID RESEARCH 12 ABBREVIATIONS 11 2 IMMUNOHISTOLOGICAL STUDY OF EXPERIMENTAL MURINE AA AMYLOIDOSIS 1 ABSTRACT 3 AMYLOID INDUCTION 4 DETECTION OF COMPONENTS OF AMYLOID FIBRILS AND MACROPHAGES 4 1 DETECTION OF F4/80 MACROPHAGES WITH LIGHT MICROSCOPY 4 2 DETECTION WITH FLUORESCENCE MICROSCOPY 4 3 DETECTION WITH CONFOCAL LASER-SCANNING MICROSCOPY 4 4 DETECTION WITH ELECTRON MICROSCOPY 5 INDUCTION OF AMYLOID DEPOSITION IN THE MARGINAL ZONE 6 TIME-KINETIC DETECTION OF AMYLOID COMPONENTS AND MACROPHAGES BY DOUBLE IMMUNO? UORESCENCE METHOD 7 FORMATION OF AMYLOID FIBRILS CONTENTS 8 RESORPTION OF AMYLOID FIBRILS 12 VISUALIZATION OF PROTEIN DEPOSITS IN VITRO 12 1 REPORTERS OF AMYLOID STRUCTURE 1 ABSTRACT 2 COMMON ELEMENTS OF AMYLOID FIBRILS 3 PROBES IN WHICH AMYLOID FIBRILS INDUCE CHANGES 3 1 PROBES OF FIBRIL FORMATION 3 2 PROBES OF SOLUBLE A? MONOMER CONFORMATION 4 TIGHT BINDING PROBES FOR AMYLOID IMAGING 4 1 DISTINCTION BETWEEN PLAQUES AND NEURO? BRILLARY TANGLES 4 2 BINDING PROTEINS 4 3 SMALL MOLECULE BINDERS 5 THE PHENOMENON OF COGNATE PEPTIDE RECOGNITION 6 CONFORMATION-DEPENDENT ANTIBODIES 6 1 CLINICAL UTILITY 6 2 CONFORMATIONAL EPITOPES 6 3 ANTI? BRILLAR ANTIBODIES 6 4 ANTIOLIGOMER ANTIBODIES 12 2 THREE-DIMENSIONAL STRUCTURAL ANALYSIS OF AMYLOID FIBRILS BY ELECTRON MICROSCOPY 1 ABSTRACT 2 INTRODUCTION: STRUCTURAL STUDIES 3 AMYLOID FIBRIL STRUCTURE AND THE CROSS-? FOLD 4 EM METHODS FOR AMYLOID 4 1 STRUCTURAL INFORMATION DEPENDS ON THE TYPE OF EM SAMPLE PREPARATION 4 2 3D RECONSTRUCTION FROM ELECTRON MICROGRAPHS 4 3 SORTING OUT STRUCTURAL VARIATIONS BY IMAGE ANALYSIS 5 RESULTS OF EM STUDIES 5 1 PROTO? LAMENT ARRANGEMENT AND FIBRIL MORPHOLOGY 5 1 1 THE ?-STRANDS IN AMYLOID FIBRILS STACK WITH A SLIGHT, LEFT-HANDED TWIST 5 2 PRION AMYLOID MODELS FROM CRYSTALLINE ARRAYS 5 3 THE STRUCTURE OF INTERMEDIATE OLIGOMERS—THE TOXIC AGENT? CONTENTS 6 PROSPECTS FOR FIBRIL STRUCTURE DETERMINATION 12 3 ATOMIC FORCE MICROSCOPY 1 ABSTRACT 3 AFM 4 STUDIES OF A? PEPTIDE AGGREGATION AND MORPHOLOGY 4 1 A? AGGREGATION AND FIBRILLIZATION 4 1 1 COMMONLY OBSERVED A? MORPHOLOGIES 4 1 2 SEEDING AND AMYLOIDOGENIC PEPTIDE FRAGMENTS 4 2 FACTORS MODULATING A? AGGREGATION 4 2 1 ROLE OF SOLUTION CONDITIONS 4 2 2 INTERACTIONS OF A? WITH LIPIDS 4 2 3 OTHER FACTORS AFFECTING A? AGGREGATION 5 STUDIES OF ?-SYN AGGREGATION AND MORPHOLOGY 5 1 ?-SYN AGGREGATION AND FIBRILLIZATION 5 1 1 FIBRIL ASSEMBLY PATHWAY 5 1 2 THE EXPLORATION OF ?-SYN SEQUENCE: STUDIES OF MUTANTS AND FRAGMENTS 5 2 FACTORS MODULATING ?-SYN AGGREGATION 5 2 1 THE ROLE OF PH 5 2 2 INTERACTIONS OF ?-SYN WITH LIPIDS 5 2 3 OTHER FACTORS AFFECTING ?-SYN AGGREGATION 6 STUDIES OF OTHER AMYLOID-FORMING PEPTIDES 7 CONCLUSIONS 8 ABBREVIATIONS 12 4 DIRECT OBSERVATION OF AMYLOID FIBRIL GROWTH MONITORED BY TOTAL INTERNAL RE? ECTION FLUORESCENCE MICROSCOPY 1 ABSTRACT 3 EXPERIMENTAL PROCEDURES 3 1 FLUORESCENCE MICROSCOPY 3 2 DIRECT OBSERVATION OF AMYLOID FIBRILS 3 3 TIME-LAPSE OBSERVATION OF AMYLOID FIBRILS 4 RESULTS AND DISCUSSION 4 1 THT OBSERVATION OF ?2-M AMYLOID FIBRILS CONTENTS 4 2 KINETICS OF FIBRIL EXTENSION 4 3 MEDIN FRAGMENT AND A?(1–40) 5 CONCLUSION 13 ANIMAL AND CELL MODELS OF HUMAN NEURODEGENERATIVE DISORDERS 13 1 DROSOPHILA AND C ELEGANS MODELS OF HUMAN AGE-ASSOCIATED NEURODEGENERATIVE DISEASES 1 ABSTRACT 3 MODELING HUMAN POLYGLUTAMINE DISEASES 3 1 HUMAN POLYGLUTAMINE DISEASES 3 2 MODELING POLYGLUTAMINE DISEASES IN DROSOPHILA MELANOGASTER 3 3 LESSONS FROM FLY MODELS: SUPPRESSORS AND ENHANCERS OF POLYQ TOXICITY 3 3 1 CHAPERONES AS SUPPRESSORS OF POLYQ TOXICITY 3 3 2 TRANSCRIPTIONAL ACTIVITY MODULATES POLYQ TOXICITY 3 3 3 PATHOGENIC POLYQ PROTEIN CAUSES AXONAL TRANSPORT DEFECTS 3 3 4 ADDITIONAL MODI? ERS OF POLYQ PATHOGENICITY 3 4 C ELEGANS MODELS OF POLYGLUTAMINE DISEASE 3 5 LESSONS FROM NEMATODE MODELS: SUPPRESSORS AND ENHANCERS 4 MODELING NONCODING TRINUCLEOTIDE REPEAT DISEASES 4 1 NONCODING TRINUCLEOTIDE REPEAT DISEASES 4 2 MODELING FRAGILE X IN THE FLY 4 3 A MODEL FOR SCA8 5 MODELING PD 5 1 PD 5 2 ?-SYNUCLEIN MODELS FOR DOMINANT PD IN THE FLY 5 3 PARKIN MODELS FOR RECESSIVE PARKINSONISM IN THE FLY 5 4 FLY MODI? ERS OF PD 5 5 MODELING PD IN C ELEGANS 6 MODELING ALZHEIMER’S AND RELATED DISEASES 6 1 ALZHEIMER’S AND RELATED DISEASES 6 2 MODELING AD WITH A? AND APP IN DROSOPHILA 6 3 TAU MODELS IN DROSOPHILA 6 4 MODI? ERS OF AD AND TAUPATHIES IN THE FLY 6 5 MODELING ALZHEIMER’S AND RELATED DISEASES IN C ELEGANS 6 6 C ELEGANS MODI? ERS OF AD PHENOTYPES 7 FUTURE DIRECTIONS CONTENTS 13 2 GENETICALLY ENGINEERED MOUSE MODELS OF NEURODEGENERATIVE DISORDERS 1 ABSTRACT 3 ALZHEIMER’S DISEASE AND CEREBROVASCULAR AMYLOIDOSIS 3 1 MODELING THE PATHOGENESIS OF AD IN ANIMALS 3 2 APP TG MODELS OF AD 3 3 BEHAVIORAL DE? CITS AND NEURODEGENERATION IN APP TG MODELS OF AD 3 4 CROSSES OF APP TGS WITH OTHER LINES 3 5 MODELS OF CEREBROVASCULAR AMYLOIDOSIS 3 6 NEURO? BRILLARY PATHOLOGY IN TG MODELS OF AD 4 FRONTO-TEMPORAL DEMENTIAS AND TAUOPATHIES 4 1 INTRODUCTION TO THE PATHOGENESIS OF TAUOPATHIES IN ANIMAL MODELS 4 2 TRANSGENIC MODELS OF NEURO? BRILLARY TANGLE DISEASE 5 LEWY BODY DEMENTIA AND PD 5 1 ROLE OF ?-SYNUCLEIN IN THE PATHOGENESIS OF LBD 5 2 ?-SYNUCLEIN TG MODELS OF LBD 5 3 MODELING OTHER ENVIRONMENTAL AND GENETIC FACTORS IN LBD TG MODELS 6 AMYOTROPHIC LATERAL SCLEROSIS 7 NEURODEGENERATIVE DISORDERS WITH TRINUCLEOTIDE REPEATS 7 1 INTRODUCTION TO THE PATHOGENESIS OF TRINUCLEOTIDE REPEAT DISORDERS IN ANIMAL MODELS 7 2 MODELS OF HD 7 3 MODELS OF NONCODING TRINUCLEOTIDE REPEAT DISORDERS 8 CONCLUSIONS STRUCTURAL AND CONFORMATIONAL PREREQUISITES OF AMYLOIDOGENESIS 1 ABSTRACT RECENT REPORTS GIVE STRONG SUPPORT TO THE IDEA THAT AMYLOID ? BRIL FORMATION AND THE SUBSEQUENT DEVELOPMENT OF PROTEIN DEPOSITION DISEASES ORIGINATE FROM CONFORMATIONAL CHANGES IN CORRESPONDING AMYLOIDOGENIC PROTEINS IN THIS REVIEW RECENT ? NDINGS ARE SURVEYED TO ILLUSTRATE THAT PROTEIN ? BRILLO- GENESIS REQUIRES A PARTIALLY FOLDED CONFORMATION THIS AMYLOIDOGENIC CONFORMATION IS RELATIVELY UNFOLDED, AND SHARES MANY STRUCTURAL PROPERTIES WITH THE PREMOLTEN GLOBULE STATE, A PARTIALLY FOLDED INTERMEDIATE FREQUENTLY OBSERVED IN THE EARLY STAGES OF PROTEIN FOLDING AND UNDER SOME EQUILIBRIUM CONDITIONS THE INHERENT ? EXIBILITY OF SUCH AN INTERMEDIATE IS ESSENTIAL IN ALLOWING THE CONFORMATIONAL REARRANGEMENTS NECESSARY TO FORM THE CORE CROSS-?; STRUCTURE OF THE AMYLOID ? BRIL 2 INTRODUCTION A SIGNI? CANT NUMBER OF HUMAN DISEASES, INCLUDING MANY NEURODEGENERATIVE DISORDERS AND THE AMYLOIDOSES, ORIGINATE FROM THE DEPOSITION OF STABLE, ORDERED, ? LAMENTOUS PROTEIN AGGREGATES, KNOWN AS AMYLOID ? BRILS IN EACH OF THESE PATHOLOGICAL STATES, A SPECI? C PROTEIN OR PROTEIN FRAGMENT CHANGES FROM ITS NATURAL SOLUBLE FORM INTO INSOLUBLE ? BRILS, WHICH ACCUMULATE IN A VARIETY OF ORGANS AND TISSUES (KELLY, 1998; DOBSON, 1999; BELLOTTI ET AL , 1999; UVERSKY ET AL , 1999B, 1999C; ROCHET AND LANSBURY, 2000; UVERSKY AND FINK, 2004) ALTHOUGH APPROXIMATELY 20 DIFFERENT PROTEINS ARE KNOWN TO BE INVOLVED IN THE AMYLOIDOSES (EXTRACELLULAR DEPOSITS), THEY ARE MOSTLY UNRELATED IN TERMS OF SEQUENCE OR STRUCTURE IN ADDITION, A NUMBER OF DISEASES ALSO INVOLVE THE DEPOSITION OF ? BRILLAR INTRACELLULAR DEPOSITS, AS WELL AS NON? BRILLAR DEPOSITS PRIOR TO ? BRILLATION, AMYLOIDOGENIC POLYPEPTIDES MAY BE RICH IN ?-SHEET, ?-HELIX, ?-HELIX, OR CONTAIN BOTH ?-HELICES AND ?-SHEETS (SEE TABLE 1-1) THEY MAY BE GLOBULAR PROTEINS WITH RIGID 3D- STRUCTURE OR BELONG TO THE CLASS OF NATIVELY UNFOLDED (OR INTRINSICALLY UNSTRUCTURED) PROTEINS DESPITE THESE DIFFERENCES, THE ? BRILS FROM DIFFERENT PATHOLOGIES DISPLAY MANY COMMON PROPERTIES INCLUDING A CORE CROSS-?-SHEET STRUCTURE IN WHICH CONTINUOUS ?-SHEETS ARE FORMED WITH ?-STRANDS RUNNING PER- PENDICULAR TO THE LONG AXIS OF THE ? BRILS (SUNDE ET AL , 1997) ALL ? BRILS HAVE SIMILAR MORPHOLOGIES, BEING TWISTED, ROPE-LIKE STRUCTURES, RE? ECTING A ? LAMENTOUS SUBSTRUCTURE AMYLOID ? BRILS HAVE BEEN FORMED IN VITRO FROM DISEASE-ASSOCIATED AS WELL AS FROM DISEASE-UNRELATED PROTEINS AND PEPTIDES (SEE TABLE 1-1) MOREOVER, THERE IS AN INCREASING BELIEF THAT THE ABILITY TO ? BRILLATE IS A GENERIC PROPERTY OF A POLYPEPTIDE CHAIN, AND ALL PROTEINS ARE POTENTIALLY ABLE TO FORM AMYLOID ? BRILS UNDER APPROPRIATE CONDITIONS (DOBSON, 1999, 2001; FANDRICH ET AL , 2001; PERTINHEZ ET AL , 2001)