neurodegeneration and prion...

NEURODEGENERATIONAND PRION DISEASE

NEURODEGENERATIONAND PRION DISEASE

Edited by

DAVID R. BROWNUniversity of Bath, UK

Library of Congress Cataloging-in-Publication Data

Neurodegeneration and prion disease / edited by David R. Brownp. ; cm.

Includes bibliographical references and index.ISBN 0-387-23922-7 (alk. paper)1. Prion Diseases. 2. Nervous system—Degeneration. I. Brown, David R., PhD.[DNLM: 1. Prion Diseases—physiopathology. 2. Cell Death—physiology. 3. NerveDegeneration—etiology. 4. Neurons—physiology. 5. Prion Diseases—complications.6. Prions—pathogenicity. WL 300 N4938152 2005]QR201.P737N48 2005616.8′3—dc22

2004062570

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To:

Lorna Jessica Hellena Brownand Hadassah Margaret Irmgard Brown

As you grow and to other things pass onYour path winding through Spring and stormAnd from treacherous heaven away you turnYou remain beloved daughters of this black swan.

David R. Brown, MSc. Ph.D.

David Brown has worked in the field of prion disease or TSEs for overten years. He was born in Australia but spent part of his early child-hood in the United Kingdom. After returning to Australia he completeshis schooling in Sydney and attended Sydney University. There he com-pleted a Bachelor of Science degree in biochemistry, a Master of Sciencedegree in neurobiology and a Doctor of Philosophy degree also in Neu-roscience. His initial research interests included neuronal growth fac-tors and topographic innervation of toad muscle. Following three yearsof postdoctoral research, Dr. Brown left Australia in 1994. Since thenhe has worked in the Albert Einstein College of Medicine in New York,the Department of Neuropathology in Gottingen and the Department ofBiochemistry at the University of Cambridge. His interest in prion beganduring his four years researching in Germany. After returning to workin the United Kingdom to work at Cambridge, Dr. Brown establishedhis own independent research group that quickly gained internationalrecognition. In parallel with research focusing on the function of the prionprotein and mechanisms of cell death in neurodegeneration, Dr. Brown’sresearch has also investigated basic aspects of cellular neurobiology in-cluding the nature of the interactions between neurones and glial cells.David Brown is currently a Reader in Biochemistry at the University ofBath and his research continues to reap international recognition andacclaim. He is also a member of the Spongiform Encephalopathy Advi-sory Committee that advises the UK government on issues to do withBSE and variant CJD.

Contents

Contributors xiEditor Bio vii

Chapters

1. IntroductionDavid R. Brown 1

2. Neuropathology of transmissible spongiformencephalopathies (prion diseases)Pawel P. Liberski and James W. Ironside 13

3. Central pathogenesis of prion diseasesUrsula Unterberger, Till Voigtlander, and Herbert Budka 49

4. Hereditary prion protein AmyloidosesBernardino Ghetti, Pedro Piccardo, Orso Bugiani,Gianluigi Forloni, Michela Morbin, Mario Salmonaand Fabrizio Tagliavini 83

5. Mouse behavioural studies and what they can teach usabout prion diseasesColm Cunningham 111

6. Electrophysiological approaches to the study ofprion diseasesNikki K. MacLeod, Alex R. Johnston and John C. Curtis 139

7. Prion protein, prion protein-like protein, andneurodegenerationSuehiro Sakaguchi 167

x Contents

8. Oxidative stress and mitochondrial dysfunction inneurodegeneration of transmissible spongiformencephalopathies (TSEs)Boe-Hyun Kim, Jae-Il Kim, Richard I. Carp,Yong-Sun Kim 195

9. Mechanisms of prion toxicity and their relationshipto prion infectivityLaura Vella, Andrew F. Hill and Roberto Cappai 217

10. A stone guest on the brain: Death as a prionDavid R. Brown 241

11. Molecular mechanisms mediating neuronal cell death inexperimental models of prion diseases, in vitroTullio Florio, Stefano Thellung and Gennaro Schettini 273

12. Processing and mis-processing of the prion protein:Insights into the pathogenesis of familial prion disordersNeena Singh, Yaping Gu, Sharmila Bose,Subhabrata Basu, Xiu Luo, Richa Mishra,and Oscar Kuruvilla 299

13. Signaling pathways controling prion neurotoxicity: Role ofendoplasmic reticulum stress-mediated apoptosisRodrigo Morales, Claudio Hetz and Claudio Soto 319

14. Cell culture models to unravel prion protein functionand aberrancies in TSEKatarina Bedecs 345

15. Insights into the cellular trafficking of prion proteinsMax Nunziante, Sabine Gilch and Hermann M. Schatzl 379

16. The molecular basis of prion protein-mediatedneuronal damageRamanujan S. Hegde and Neena S. Rane 407

17. Conclusion: Intervention, the final frontierDavid R. Brown 451

Index 457

List of Contributors

Subhabrata BasuInstitute of Pathology, Case Western Reserve University, 2085,Adelbert Road, Cleveland, Ohio, USA.

Katarina BedecsDepartment of Biochemistry and Biophysics, Stockholm University,Svante Arrhenius vag 12, S-10691 Stockholm, Sweden.

Sharmila BoseDepartment of Molecular Cellular & Developmental Biology,The University of Michigan, 830 North University Avenue,Ann Arbor, Michigan, USA.

David R. BrownDepartment of Biology and Biochemistry, University of Bath,Bath, BA2 7AY, UK.

Herbert BudkaInstitute of Neurology, Medical University of Vienna, AKH 4J,Wahringer Gurtel 18-20, A-1097 Vienna, Austria.

Orso BugianiIstituto Nazionale Neurologico “Carlo Besta”, Division ofNeuropathology and Biochemistry and Molecular Pharmacology,Via Celoria 11, 20133 Milan, Italy

Roberto CappaiDepartment of Pathology and The Centre for Neuroscience,The University of Melbourne,Melbourne, Victoria 3010. Australia.

Richard I. CarpNew York State Institute for Basic Research in DevelopmentalDisabilities, 1050 Forest Hill Road, Staten Island, NY 10314, USA.

xii List of Contributors

John C. CurtisBiomedical Sciences, Hugh Robson Building, George Square,Edinburgh, EH8 9XD, UK.

Colm CunninghamCNS Inflammation Group, School ofBiological Sciences, Biomedical Sciences Building, University ofSouthampton, Southampton SO16 7PX, UK.

Tullio FlorioSection of Pharmacology, Dept. Oncology Biology and Genetics,University of Genova, Genova, Italy and Pharmacology andNeuroscience, National Institute for Cancer Research (IST)Genova, Italy.

Gianluigi ForloniIstituto di Ricerche Farmacologiche “Mario Negri”, Department ofNeuroscience, Via Eritrea 62, 20157 Milan, Italy

Bernardino GhettiIndiana University School of Medicine, Department of Pathology andLaboratory Medicine, 635 Barnhill Drive MSA138, Indianapolis,IN 46202, USA

Sabine GilchInstitute of Virology Prion Research Group, TechnicalUniversity of Munich, Biedersteinerstrasse 29,80802 Munich, Germany.

Yaping GuInstitute of Pathology, Case Western Reserve University, 2085,Adelbert Road, Cleveland, Ohio, USA.

Ramanujan S. HegdeCell Biology and Metabolism Branch, NICHD, National Institutes ofHealth, 18 Library Drive, Bldg. 18T, Room 101, Bethesda,MD 20892, USA.

Claudio HetzInstituto de Ciencias Biomedicas, Universidad de Chile,Santiago, Chile.

Andrew F. HillDepartment of Biochemistry and Molecular Biology,and Department of Pathology and The Centre for Neuroscience,The University of Melbourne, Melbourne,Victoria 3010, Australia.

List of Contributors xiii

James W. IronsideNational Creutzfeldt-Jakob Disease Surveillance Unit,Western General Hospital, Edinburgh, EH4 2XU, UK.

Alex R. JohnstonBiomedical Sciences, Hugh Robson Building, George Square,Edinburgh, EH8 9XD, UK.

Boe-Hyun KimIlsong Institute of Life Science, and bDepartment of Microbiology,College of Medicine, Hallym University, Ilsong Building,Kwanyang-dong 1605-4, Dongan-gu, Anyang 431-060,South Korea.

Jae-Il KimNew York State Institute for Basic Research in DevelopmentalDisabilities, 1050 Forest Hill Road, Staten Island, NY 10314, USA.

Yong-Sun KimIlsong Institute of Life Science, and Department of Microbiology,College of Medicine, Hallym University, Ilsong Building,Kwanyang-dong 1605-4, Dongan-gu, Anyang 431-060, South Korea.

Oscar KuruvillaInstitute of Pathology, Case Western Reserve University, 2085,Adelbert Road, Cleveland, Ohio, USA.

Pawel P. LiberskiLaboratory of Electron Microscopy and Neuropathology,Department of Molecular Pathology and Neuropathology, MedicalUniversity, Lodz, Poland.

Xiu LuoInstitute of Pathology, Case Western Reserve University, 2085,Adelbert Road, Cleveland, Ohio, USA.

Nikki K. MacLeodBiomedical Sciences, Hugh Robson Building, George Square,Edinburgh, EH8 9XD, UK.

Rodrigo MoralesDepartment of Neurology, University of Texas Medical Branch,Galveston, Texas, USA.

Michela MorbinIstituto Nazionale Neurologico “Carlo Besta”, Division ofNeuropathology and Neurology 5, Via Celoria 11, 20133 Milan, Italy

xiv List of Contributors

Richa MishraInstitute of Pathology, Case Western Reserve University, 2085,Adelbert Road, Cleveland, Ohio, USA.

Max NunzianteInstitute of Virology Prion Research Group, Technical Universityof Munich, Biedersteinerstrasse 29, 80802 Munich, Germany.

Pedro PiccardoCenter for Biologics Evaluation and Research, Food and DrugAdministration, Rockville, MD 20852, USA and Indiana UniversitySchool of Medicine, Department of Pathology and LaboratoryMedicine, 635 Barnhill Drive MSA138, Indianapolis, IN 46202, USA

Neena S. RaneCell Biology and Metabolism Branch, NICHD, National Institutes ofHealth, 18 Library Drive, Bldg. 18T, Room 101, Bethesda,MD 20892, USA.

Suehiro SakaguchiDepartment of Molecular Microbiology and Immunology, NagasakiUniversity Graduate School of Biomedical Sciences, Sakamoto 1-12-4,Nagasaki 852-8523 and PRESTO, Japan Science and TechnologyAgency, 4-1-8 Honcho Kawaguchi, Saitama, Japan.

Mario SalmonaIstituto di Ricerche Farmacologiche “Mario Negri”, Department ofBiochemistry and Molecular Pharmacology, Via Eritrea 62, 20157Milan, Italy

Hermann M. SchatzlInstitute of Virology Prion Research Group, Technical Universityof Munich, Biedersteinerstrasse 29, 80802 Munich, Germany.

Gennaro SchettiniSection of Pharmacology, Dept. Oncology Biology and Genetics,University of Genova, Genova, Italy and Pharmacology andNeuroscience, National Institute for Cancer Research (IST)Genova, Italy.

Neena SinghInstitute of Pathology, Case Western Reserve University, 2085,Adelbert Road, Cleveland, Ohio, USA.

Claudio SotoDepartment of Neurology, University of Texas Medical Branch,Galveston, Texas, USA.

List of Contributors xv

Fabrizio TagliaviniIstituto Nazionale Neurologico “Carlo Besta”, Division ofNeuropathology and Neurology 5, Via Celoria 11, 20133 Milan, Italy

Stefano ThellungSection of Pharmacology, Dept. Oncology Biology and Genetics,University of Genova, Genova, Italy.

Ursula UnterbergerInstitute of Neurology, Medical University of Vienna, AKH 4J,Wahringer Gurtel 18-20, A-1097 Vienna, Austria.

Laura VellaDepartment of Biochemistry and Molecular Biology, and Departmentof Pathology, The University of Melbourne, Melbourne,Victoria 3010, Australia.

Till VoigtlanderInstitute of Neurology, Medical University of Vienna, AKH 4J,Wahringer Gurtel 18-20, A-1097 Vienna, Austria.

Chapter 1

INTRODUCTION

David R. Brown

Department of Biology and Biochemistry,University of Bath, Bath BA2 7AY, UK

In 1982 Stanley Prusiner and colleagues purified an abnormal proteinfrom the brains of mice experimentally infected with a rare sheep dis-ease called scrapie1. This protein was called the prion protein. Earlierwork had suggested that this diseases and others, loosely collected to-gether as transmissible spongiform encephalopathies (TSEs), were nottransmitted by conventional infectious agents. Prusiner suggested thatthis new protein was the infectious agent in these diseases2. Such acontentious suggestion lead to ferocious debate. Many researchers stillmaintained that there was no such thing as an infectious protein. De-spite this, by 1990 most people accepted that the cause of the TSEs wasthe abnormal isoform of the prion protein his research group had iden-tified. The most convincing evidence for this had come from the workof Charles Weissmann, whose prion protein knockout mice could notbe infected because they lacked expression of the protein that was nowforever linked to these disease3,4. Since then it has become more widelyaccepted for these diseases to be termed prion diseases. In 1997 whenStanley Prusiner won the Nobel Prize for his work on prion diseases5.Even then, there was still an element of resistance in the scientific com-munity. It was considered that, in order the transmissible agent to trulybe a protein only, the protein would have to be generated from a recom-binant source.

In 2004 that evidence emerged. Recombinant protein injected intomice led to a prion disease that could then be transmitted to other mice6.Naturally, scepticism still continues about this novel theory. Those whowork in the prion field know that this is simply part of the game. Intense

2 Brown

scepticism of any findings on any aspect of research in prion diseasesmakes progress in the field very slow. Part of the problem is probablydue to a fundamental misunderstanding of the nature of prion diseases.Although the diseases can be transmitted experimentally, prion diseasesare not contagious diseases as are bacterial or viral disease.

Some forms of prion diseases are inherited, such as Gerstmann-Straussler-Scheinker syndrome (GSS)7–8. GSS is linked to point muta-tions in the prion protein gene (prnp). Despite inheriting what amounts toa dominant lethal genetic mutation, GSS patients usually reach their fifthdecade of life before symtoms of the disease emerge. This clearly linksonset of these diseases to something inherant in the normal aging pro-cess. Other forms of transmission have occurred because of human in-tervention. Iatrogenic CJD results from use of tissues or hormone deriva-tives of tissue from people who had the more conventional disease,sporadic CJD. Kuru, a disease of the native of New Guinea occurredbecause of the ritual practice of eating brains from older relatives9. Thenew disease, variant CJD had been linked to the eating of food contam-inated with the BSE agent. BSE (bovine spongiform encephalopathy)and variant CJD share many similarities and the two diseases clearlyhave a similar origin10–11. It is widely accepted that BSE caused vCJDbut there is also considerable doubt that vCJD arose from the eatingof BSE contaminated meat. BSE was also mostly the result of humanintervention. The feeding of rendered animal remains back to dairy cat-tle result in tens of thousands of cases of BSE carrying cows. Yet, nowthat this practice is banned and BSE number have dropped dramatically,there remains a significant number of BSE cases. The cause of thesecases of BSE remains unknown. This is similar to the majority of casesof human prion disease. The major form of human prion diseases issporadic CJD. This disease cannot be linked to any form of infection.Similarly, the disease of sheep called scrapie can also not be linked toany specific infection event. Scrapie is the first described prion diseasewith reports dating back to the 15th century.

As panic over the BSE epidemic subsides and the predicted expo-nential increase in variant CJD cases has not happened, more rationalthought has entered into the arena to assess the possible cause of themajor forms of prion diseases. The two logical explanations that havebeen put forward are the following: The sporadic forms of prion diseasescould arise through a freak event in the normal ageing process. As men-tioned above, GSS does not manifest until late in life. This implies that thekinetics of prion formation are very slow and take upwards of 30 years toresult in abnormal prion protein forming in the brain. Alternatively somechange that occurs as we grow older may be needed to trigger proteinconversion to make the normal cellular form of the prion protein to flip

Introduction 3

conformation and generated PrPSc. Once a significant amount of thisprotein is formed it is able to catalyse its own conversion and sponta-neous deposition of large amounts of PrPSc results in the pathologicalchanges leading to CJD12. The possible change in the ageing brain isthe gradual decay in the balance between oxidative damage and an-tioxidant defence. The second hypothesis about the cause of sporadicprion diseases is that exposure to an agent in the environment may trig-ger protein conversion. Evidence for this comes from the existence oflocalised hot spots for different prion diseases13. The disease of deer,chronic wasting disease, is very heavily localised to small areas withUS states such as Colorado. In Iceland, some farms have recurrentscrapie problems while other remain consistently scrapie free. Whatin the environment could have such an effect remains to be verified.However, the prime candidate has been manganese14. Manganese ac-cumulates in the brains of patients and animals with prion diseases15–16.However, it remains to be shown whether manganese is causative, a fac-tor that enhances the incidence of an ever present disease or is simplycoincidental.

The implication of all the forgoing is that prion diseases are funda-mentally a result of a normal brain protein becoming conformationallyaltered. An event or a series of similar events result in the stabilisationin a conformational switch in the isoform of the protein generated bythe brain12. The trigger of this can be one of three possibilities. The firstis the introduction into the cell of preformed PrPSc aggregates. This isthen able to catalyse conformation alteration of prion protein generatedby that cell. The second possibility is that the normal cellular isoform ofthe prion protein (PrPc) encounters a different agent which then catal-yses conversion. This could be interaction with a metal that does notnormally bind to the protein such as manganese17. Lastly, conversionto the abnormal isoform occurs naturally but with a low probability. Thisimplies that, the kinetic equilibrium does not favour PrPSc formation butthat in time a small amount will form that is the sufficient to catalysefurther conversion by the first mechanism. An alternative version of thislast hypothesis is that PrPSc is formed in the brain all the time but mech-anism are in place which rapidly clear it away before it can autocatalysefurther PrPSc formation. Disease develops when this corrective processfalters, possibly as a result of ageing.

Understanding the common threads in all these theories and the clearlink between disease progress and expression of the prion protein andthe conformation it assumes makes discussion of any “contagion” caus-ing these disease seem absurd. Panic among both the lay and the sci-entific communities about the inherent infectiousness of prion disease ispurely an hysterical response to misinformation or wanton ignorance of

4 Brown

the fundamental truth of the cause of these disease. A normal brain gly-coprotein becomes converted to a protease resistant isoform, by what-ever mechanism, and initiates a series of pathological changes in thebrain resulting in death. This implies that to understand these diseaseswe must know what causes the patient’s own prion protein to changeconformation and understand how production of this abnormal confor-mation relates to the pathological changes that cause the death of thepatient.

Until recently study of the prion protein has focussed on PrPSc andthe normal form of the protein has been overlooked. However, the genefor PrP was first described in 198618, the year that BSE first emerged.The protein is a glycoprotein anchored to the outside of the cell by aglycosylphophatidylinositol (GPI) anchor19. This means that the proteinis attached to the membrane by a sugar group. Although there has beensome speculation about there being a transmembrane form of the pro-tein, this has largely been dismissed20–22. Similar reports of dimericforms of the protein and subcellular localisation of the protein to thecytosol or nucleus are isolated and unconfirmed23–25. The protein ishighly expressed by neurones and is concentrated in the synapse26.The expression of the protein is not specific to neurones and low levelexpression can be detected in many cell types. The age of the cellularprion protein began in 1995 with the first suggestions that a fragmentof the protein could bind copper27. This largely was ignored until 1997when a number of colleagues and I provided the first accepted evidencethat the protein binds copper in vivo28. Since then there has been over-whelming support for the idea that PrPc is a metalloprotein.

The function of the protein has been the subject of a number ofinvestigations. Despite numerous different approaches the emergingconsensus is that lack of expression of PrPc causes cells to respondpoorly to stress29. These changes can range from altered electrophysi-ological parameters30, altered sleep patterns31, modified cell adhesioncharacteristics32 and disturbed cell signalling pathways33. More sub-stantial evidence points to PrPc being some form of antioxidant. Myown research has suggested that PrPc is a molecule with the abilityto clear away superoxide radicals that would otherwise damage cellcomponents34. This would make a PrPc superoxide dismutase. Alterna-tive research has shown that PrPc can alter copper uptake into cells35

and that binding of copper to PrPc is important to the mechanism bywhich it is internalised from the cell surface36. These theories are notcontradictory, as sequesting copper is in itself an antioxidant effect.Copper has the potential to generating molecules that can cause ox-idative stress. Therefore the leading theory as to the function of PrPc isthat it is an antioxidant.

Introduction 5

Conversion of PrPc to PrPSc results in the loss of function of the proteinwithout the loss of its expression. In prion protein knockout mice, lackof expression of PrPc could be compensated for by rapid changes inexpression of other proteins that could perform similar functions. As apotential antioxidant, such rapid compensation is very likely consideringthe wide range of antioxidants that the body can mobilise and the highinduciblity of many cellular antioxidants. One implication is that loss ofPrPc function could expose neurones to assaults that cause initiate celldeath. Therefore understanding the function of this protein and how tocompensate for it could be one possible way to counteract the cell deaththat occurs in prion disease.

Prion diseases are neurodegenerative conditions. They result in avery rapid loss of neurones in specific areas of the brain. This neuronalloss occurs late in the disease and corresponds to onset of neurologicaland behavioural symptoms. In experimentally induced prion disease,there is a long incubation time between challenge with the prion dis-ease agent and the onset of symptoms. In the case of BSE this can beyears. Following onset of symptoms death from complications followsvery rapidly. In humans with CJD this can be a matter of months. If celldeath could be stopped then possibly, the CJD patient could recover.Clearly, knowing what causes this cell death is central to understandingthese disease. Surprisingly, until recent years, there has been little re-search on the mechanism of cell death in prion disease. Reviews on thesubject of “neurodegeneration and prion disease” often failed to mentionmechanisms of cell death in any detail.

The first models of the mechanism of neurodegeneration emergedfrom cell cultures studies in the early 1990s. These models showedthat PrPSc or a peptide derivative could kills neurones by an apoptoticmechanism37–38. The first finding was that was of any significance wasthat neuronal cell death requires the expression of PrPc by the targetcell39. My research was the first to show that neurones from prion proteinknockout mice were resistant to toxic prions. This was later confirmedin animal models40–41.

Advancement in the field of neurodegeneration and prion diseaseshas resulted in the research described in the chapters of this book. Thiscompilation therefore reflects the great strides that have been madein recent years. Many individual and complementary approaches havebeen taken, providing a wealth of information that has the potential toone day provide us with a possible way forward in finding preventativetreatments to halt the advance of neurodegeneration. Prion diseasesare rare but so are reliable models of most human neurodegenera-tive diseases. In this regard prion diseases are the exception as ex-periment infection of mice provides us with an accurate and essential

6 Brown

tool for research. The implication of this is that study of prion dis-eases might provide insights into neurodegeneration that are relevantto other diseases like Alzheimer’s disease where animal models don’texist.

The main model used by researchers to study prion diseases is themouse model using mouse passaged scrapie. Some researchers alsouse hamsters but the availability of transgenic mice makes mice a moreattractive choice. Studies with such mice have lead to a whole range ofinteresting research. The sheep disease scrapie can be divided into aseries of strains or different forms. These strains retain a range of char-acteristics when used to infect mice of a similar genetic background.These characteristics include the length of time the animal takes tofall ill (incubation time), localisation within the brain of pathology andextent and localisation PrPSc deposition as well as the ratio betweenthe amounts of the three glycoforms (di-, mono-, or non-gycosylated)of PrPSc deteccted42. Challenge with the scrapie agent is usually per-formed either by force feeding mice scrapie agent laced food (oral chal-lenge) or direct injection of the agent into the brain. Oral challenge isa less successful route of infection but it has provided insight into themechanism of oral transmission of prion disease.

In terms of the study of neurodegeneration, the mouse model hasproved a difficult one to provide mechanism of action. This is becauseit is difficult to separate the cause of neuronal death from the necessityto introduce PrPSc into the brain from an external source. Apoptotic celldeath occurs in the brain and this is proceeded by the activation of mi-croglia and occurs in parallel with increased astrogliosis43–46. Very earlychanges to neurones can be detected such as loss of dendritic spines47.Use of conditional prion protein knockout mice has shown that stoppingexpression of PrPc during the disease progress, after considerable PrPSc

has been formed, results in sessation of cell death and recovery of theanimal41. This really just confirms what was first identified in 1994 usingcell culture models39. Namely, that PrPc expression is necessary by thetarget cell and without it toxic prions cannot kill neurones.

Five chapters in this edition deal with rodent models. The first is theinsightful examination of changes to behavior in mice carrying scrapieby Colm Cunningham. The second by Nikki Macleod and colleaguesdescribes how electrophysiological techniques have been used to in-vestigate changes, both as a result of transgenic manipulation, and byscrapie infection. Next, Suehiro Sakaguchi discusses how transgenicmice were used to investigate prion diseases. Herbert Budka and col-leagues discuss the use of mouse models to investigate pathologicaland biochemical changes associated with neurodegeneration in priondisease. Finally, Yong-Sun Kim and colleagues discuss the possible role

Introduction 7

of oxidative stress and mitochondrial dysfunction in the cause of celldeath in prion disease based on their own research using scrapie in-fected rodents.

Another way of studying neurodegeneration without resorting to mod-els is to examine the pathological changes associated with neurodegen-eration. This requires a very thorough study of those changes in bothhuman and animal disease. A very thorough study of the pathology ofprion diseases is present in a chapter by Pawel Liberski and James Iron-side. In another study, Bernardino Ghetti and colleagues, discusses thepathology of inherited forms of prion diseases.

The remaining chapters concentrate on in vitro models. KatarinaBedecs describes the use of scrapie infected cell lines and the wayin which they provide insights into changes in the metabolism of cellsconstituatively generating PrPSc. Models of the toxic actions of PrPSc

and related peptides are discussed by a number authors. This reflectsthat this system for studying prion diseases has been in use for over tenyears and is one of the easiest to set up and provides results in a matterof weeks rather than years. Studies with scrapie infected mice can takevery long because the incubation time is usually around four months.Studies with cattle or sheep take even longer as the incubation periodfor the disease can be between two and five years. I have provided achapter summarising the vast body of work produce by my own groupsince I first began using cell culture models in 1993. Gennaro Schettini,Neena Singh, Roberto Cappai and their colleagues provide the nextthree chapters also based on work within vitro toxicity models.

Other factors can also contributed to cell survival and the onset ofprion disease. The recent work of Claudio Soto investigating the role ofendomplasmic reticulum (ER) stress in creating a cellular environmentthat would favour cell death is presented in a chapter from his group. Al-though most PrPc is anchored to the surface by a glycosylphosphatidyli-nositol anchor, it has been suggested that some amount of the proteincould be incorporated into the cell membrane. A putative stop transferelement was described many years ago and at the same time it wassuggested that the hydrophobic domain of the protein could potentialbe a transmembrane domain48. Ramanujan Hegde presents his inter-esting work that proposes that transmembrane forms of PrP could beinvolved in neurodegeneration in prion diseases. In particular this mech-anism has been suggested to be relevant for inherited forms of thesediseases.

In the final chapter Hermann Schatzl and colleagues present their el-egant and compelling work examining the mechanism of internalisationof the prion protein and the possible effect of prion mutations might haveon cell survival.

8 Brown

The broad range of the chapters indicates the great expanse of imag-inative directions taken to shine some light into darkness that surroundsthis enigmatic field. These various approaches high light the complexityof the subject and the need for continued, international and comprehen-sive support for the research of the many laboratories dedicated to finda way forward in determining the nature of neurodegeneration in priondisease. Perhaps, there is a long way to go until we develop effectivetreatments for these disorders, but the science of neurodegenerationand prion diseases has well and truly solidified into a vibrant and com-pelling field of study.

References

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2. S. B. Prusiner, Novel proteinaceous infectious particles cause scrapie. Science 216,136–144 (1982).

3. H. Bueler, Fischer, Y. Lang, H. Bluethmann, H.-P. Lipp, S. J. DeArmond, S. B. Prusiner,M. Aguet and C. Weissmann, Normal development and behaviour of mice lackingthe neuronal cell-surface PrP protein. Nature, 356, 577–582, (1992).

4. H . Bueler, A. Aguzzi, A. Sailer, R. A. Greiner., P. Autenried, M. Aguet and C.Weissmann, Mice devoid of PrP are resistant to scrapie. Cell 73, 1339–47 (1993).

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6. G. Legname, I. V. Baskakov, H. O. Nguyen, D. Riesner, F. E. Cohen, S. J. DeArmondand S. B. Prusiner, Synthetic mammalian prions. Science. 305, 673–676 (2004).

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8. D. R. Brown, Molecular advances in understanding inherited prion diseases. Mol.Neurobiol. 25, 287–302 (2002).

9. D. C. Gajdusek and C. J. Gibbs Jr. Transmission of two subacute spongiform en-cephalopathies of man (Kuru and Creutzfeldt-Jakob disease) to new world monkeys.Nature 230, 588–591 (1971).

10. J. Collinge, Human prion diseases and bovine spongiform encephalopathy (BSE).Hum. Mol. Genet. 6 1699–1705 (1997).

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Introduction 9

C. J. Transmissions to mice indicate that ‘new variant’ CJD is caused by the BSEagent. Nature, 389, 498–501 (1997).

12. F. E. Cohen and S. B. Prusiner, Pathologic conformations of prion proteins. Annu.Rev. Biochem., 67, 793–819 (1998).

13. M. Purdey, Ecosystems supporting clusters of sporadic TSEs demonstrate excessesof the radical generating divalent cation manganese and deficiencies of antioxidantco-factors; Cu, Se, Fe, Zn. Does a foreign cation substitution at PrP’s Cu domaininitiate TSE? Med. Hypoth. 54, 278–306 (2000).

14. D. R. Brown, BSE did not cause variant CJD: an alternative cause related topost-industrial environmental contamination, Medical Hypotheses, 57, 555–560(2001).

15. B.-S. Wong, S. G.Chen, M. Colucci, Z. Xie, T. Pan, T. Liu, R. Li, P. Gambetti, M.-S. Syand D. R. Brown, Aberrant metal binding by prion protein in human prion disease.J. Neurochem. 78, 1400–1408 (2001).

16. A. M. Thackray, R. Knight, S. J. Haswell, R. Bujdoso, and D. R. Brown, Metal im-balance and compromised antioxidant function are early changes in prion disease.Biochem J, 362, 253–258 (2002).

17. D. R. Brown, F. Hafiz, L. L. Glasssmith, B.-S. Wong, I. M. Jones, C. Clive, and S.J. Haswell, Consequences of manganese replacement of copper for prion proteinfunction and proteinase resistance. EMBO J. 19, 1180–1186 (2000).

18. K. Basler, B. Oesch, M . Scott, D. Westaway, M. Walchli, D. F. Groth, M. P. McKinley,S. B. Prusiner and C. Weissmann, Scrapie and cellular PrP isoforms are encodedby the same chromosomal gene. Cell 46, 417–28 (1986).

19. N. Stahl, D. R. Borchelt and S. B. Prusiner, Differential release of cellular and scrapieprion protein from cellular membranes of phosphatidylinositol specific phospholi-pase C. Biochemistry 29, 5405–5412 (1990).

20. R. S. Hegde, J. A. Mastrianni, M. R. Scott, K. D. Defea, P. Tremblay, M.Torchia, S. J. DeArmond, S. B. Prusiner, and V. R. Lingappa, A transmembraneform of the prion protein in neurodegenerative disease. Science 279, 827–834(1998).

21. R. S. Stewart and D. A. Harris, Most pathogenic mutations do not alter the membranetopology of the prion protein. J. Biol. Chem. 276, 2212–2220 (2000).

22. A. Holme, M. Daniels, J. Sassoon, and D. R. Brown, A novel method of generat-ing neuronal cell lines from gene-knockout mice to study prion protein membraneorientation. Eur. J. Neurosci. 18, 571–579 (2003).

23. R. K. Meyer, A. Lustig, B. Oesch, R. Fatzer, A. Zurbriggen, and M. Vandevelde,A monomer-dimer equilibrium of a cellular prion protein (PrPC) not observed withrecombinant PrP. J Biol Chem. 275, 38081–38087 (2000).

10 Brown

24. X. Roucou, Q. Guo, Y. Zhang, C. G. Goodyer, A. C. LeBlanc, Cytosolic prion proteinis not toxic and protects against Bax-mediated cell death in human primary neurons.J. Biol. Chem. 278, 40877–40881 (2003).

25. A. Mange, C. Crozet, S. Lehmann, F. Beranger, Scrapie-like prion protein is translo-cated to the nuclei of infected cells independently of proteasome inhibition andinteracts with chromatin. J. Cell Sci. 117, 2411–2416 (2004).

26. N. Sales, K. Rodolfo, R. Hassig, B. Faucheux, L. Di Giamberdino, and K. L. Moya,Cellular prion protein localization in rodent and primate brain. Eur. J. Neurosci.10,2464–2471 (1998).

27. M. P. Hornshaw, J. R. McDermott and J. M. Candy, Copper binding to the N-terminaltandem repeat regions of mammalian and avian prion protein. Biochem BiophysRes Commun 207, 621–629 (1995).

28. Brown D. R., Qin K., Herms J. W., Madlung A., Manson J., Strome R., FraserP. E., Kruck T., von Bohlen A., Schulz-Schaeffer W., Giese A., Westaway D. andKretzschmar H. (1997) The cellular prion protein binds copper in vivo. Nature 390,684–687.

29. D. R. Brown, R. St. J. Nicholas, and L. Canevari, Lack of prion protein expressionresults in a neuronal phenotype sensitive to stress. J. Neurosci. Res. 67, 211–224(2002).

30. J. Collinge, M. A. Whittington, K. C. Sidle, C. J. Smith, M. S. Palmer, A. R. Clarkeand J. G. Jefferys, Prion protein is necessary for normal synaptic function. Nature370, 295–297 (1994).

31. R. Huber, T. Deboe and I. Tobler Sleep deprivation in prion protein deficient micesleep deprivation in prion protein deficient mice and control mice: genotype depen-dent regional rebound. Neuroreport. 13, 1–4 (2002).

32. E. Graner, A. F. Mercadante, S. M. Zanata, V. R. Martins, D. G. Jay, R. R. Brentani,Laminin-induced PC-12 cell differentiation is inhibited following laser inactivation ofcellular prion protein. FEBS Lett. 482, 257–260 (2000).

33. L. B. Chiarini, A. R. Freitas, S. M. Zanata, R. R. Brentani, V. R. Martins, R. Linden,Cellular prion protein transduces neuroprotective signals. EMBO J. 21, 3317–26(2002).

34. D. R. Brown, B.-S. Wong, F. Hafiz, C. Clive, S. J. Haswell and I. M. Jones, Normalprion protein has an activity like that of superoxide dismutase. Biochem J 344, 1–5(1999).

35. D. R. Brown, Prion protein expression aids cellular uptake and veratridine-inducedrelease of copper. J. Neurosci. Res. 58, 717–725 (1999).

36. P. C. Pauly and D. A. Harris, Copper stimulates endocytosis of the prion protein. J.Biol. Chem. 273, 33107–33110 (1998).

Introduction 11

37. W. E. Muller, H. Ushijima, H. C. Schroder, J. M. Forrest, W. F. Schatton, P. G. Rytik, M.Heffner-Lauc, Cytoprotective effect of NMDA receptor antagonists on prion protein(PrionSc)-induced toxicity in rat cortical cell cultures. Eur. J. Pharmacol. 246, 261–267 (1993).

38. G. Forloni, N. Angeretti, R. Chiesa, E. Monzani, M. Salmona, O. Bugiani and F.Tagliavini, Neurotoxicity of a prion protein fragment. Nature 362, 543–546 (1993).

39. D. R. Brown, J. Herms, and H. A. Kretzschmar, Mouse cortical cells lacking cellularPrP survive in culture with a neurotoxic PrP fragment. Neuroreport 5, 2057–2060(1994).

40. S. Brandner, S. Isenmann, A. Raeber, M. Fischer, A. Sailer, Y. Kobayashi, S. Marino,C. Weissmann and A. Aguzzi, Normal host prion protein necessary for scrapie-induced neurotoxicity. Nature, 379, 339–343 (1996).

41. G. Mallucci, A. Dickinson, J. Linehan, P. C. Klohn, S. Brandner and J. Collinge,Depleting neuronal PrP in prion infection prevents disease and reverses spongiosis.Science. 302, 871–874 (2003).

42. M. E. Bruce and H. Fraser Scrapie strain variation and its implications. Curr. Top.Microbiol. Immunol., 172, 125–138 (1991).

43. A. E. Williams, L. J. Lawson, V. H. Perry, and H. Faser, Characterization of microglialresponse in murine scrapie. Neuropathol. Appl. Neurobiol., 20, 47–55 (1994).

44. A. Giese, M. H. Groschup, B. Hess, and H. A. Kretzschmar, Neuronal cell death inscrapie-infected mice is due to apoptosis. Brain Pathol. 5, 213–221 (1995).

45. S. Betmouni, V. H. Perry, and J. L. Gordon, Evidence for an early inflammatoryresponse in the central nervous system of mice with scrapie. Neuroscience, 74,1–5 (1996).

46. A. Giese, D. R. Brown, M. H. Groschup, C. Feldmann, I. Haist and H. A. Kretzschmar,(1998) Role of microglia in neuronal cell death in prion disease. Brain Pathol. 8,449–457.

47. P. V. Belichenko, D. Brown, M. Jeffrey and J. R. Fraser, Dendritic and synaptic al-terations of hippocampal pyramidal neurones in scrapie-infected mice. NeuropatholAppl Neurobiol. 26, 143–149 (2000).

48. C. S. Yost, C. D. Lopez, S. B. Prusiner, R. M. Myers, and V. R. Lingappa, Non-hydrophobic extracytoplasmic determinant of stop transfer in the prion protein.Nature 343, 669–672 (1990).

Chapter 2

NEUROPATHOLOGY OF TRANSMISSIBLESPONGIFORM ENCEPHALOPATHIES(PRION DISEASES)

Pawel P. Liberski1 and James W. Ironside2

1Laboratory of Electron Microscopy and Neuropathology,Department of Molecular Pathology and Neuropathology,Medical University, Lodz, Poland2National Creutzfeldt-Jakob Disease Surveillance Unit,Western General Hospital, Edinburgh, EH4 2XU, UK

2.1. Introduction

The transmissible spongiform encephalopathies (TSEs) or priondiseases are a group of neurodegenerative disorders which includekuru1, Creutzfeldt-Jakob disease (CJD)2, variant Creutzfeldt-Jakob dis-ease (vCJD), Gerstmann-Straussler-Scheinker (GSS) disease3, andfatal familial insomnia4 in man, natural scrapie in sheep, goats andmufflons, transmissible mink encephalopathy in ranch-reared mink5,chronic wasting disease of mule deer and elk in the USA6 and Canada,bovine spongiform encephalopathy (BSE) or “mad cow disease”7 and itsanalogues in several exotic species of antelopes and wild felids in zoo-logical gardens and feline spongiform encephalopathy in domestic cats.

These disorders are caused by a still not completely understoodpathogen variously referred to as a “prion” (predominantly) or a slow,unconventional or atypical virus, or “virino” (rarely). Despite wide accep-tance for the prion theory, these names still reflect different views aboutthe true molecular structure of the pathogen and, by the same token, ourignorance of its nature. Those who prefer to view this pathogen as com-posed “predominantly or exclusively” of a pathologically folded protein

14 Liberski and Ironside

(PrPSc; Sc from scrapie or PrPd; d from disease), use the term “prion”;hence the term “prion diseases”.

The “virino” hypothesis suggests that the pathogen is a molecularchimera composed of a still-to-be-discovered nucleic acid and a shell-protein which is host-encoded (perhaps PrPd). The virus hypothesissimply suggests that the pathogen is a yet-to-be-identified unconven-tional virus. The “unified theory” of Weissmann8, not unlike the virinotheory, suggests that the agent is a molecular chimera of the misfoldedprotein that confers infectivity and an unidentified oligonucleotide thatspecifies strain characteristics.

2.2. Nomenclature

The nomenclature of PrP species is confusing. PrPc is a normalcellular isoform. PrPSc (PrPres or PrPd, from disease) is a patholog-ical misfolded protein. PrPSc is operationally defined as resistant toproteinase K (PK) and insoluble in denaturing detergent; however, insome diseases, pathological isoform of PrP is not PK resistant9. Thus,we prefer to use the neutral term PrPd which denotes that misfoldedspecies of PrP which is disease-associated; PK-resistant or not. PrP27-30 is a proteolytic cleavage product of PrPd which is sometimesreferred to as PrPres (res from resistant) when generated following in-complete proteolytic digestion in Western blotting.

2.3. PrP, its gene, the ‘‘prion” hypothesis

PrPc is a highly conserved sialoglycoprotein encoded by a cellulargene mapped to chromosome 20 in man and 2 in mouse10. The geneis ubiquitous; it has been cloned in numerous mammalian species in-cluded marsupials and there are analogues of this gene in birds, rep-tiles, amphibians, and recently fish; those in Drosophila and nematodesappeared to be cloning artefacts. PrP 27-30 was first discovered as aprotein co-purifying with infectivity in extracts derived from brains in-fected with the 263K strain of scrapie agent which led to the conclusionthat PrP is a part of infectivity.

The “prion” hypothesis, which is deeply rooted in this associationbetween PrP and infectivity, was formulated by Stanley B. Prusinerin 198211. The hypothesis postulated that the scrapie agent was aproteinaceous infectious particle, because infectivity was dependent onprotein but resistant to methods known to inactivate nucleic acids. A simi-lar proposal had been presented a decade earlier by many investigatorswho all developed the earlier suggestion based on irradiation studies,that scrapie agent was devoid of disease-specific nucleic acid12.

Neuropathology of Transmissible Spongiform Encephalopathies 15

Like many amyloid proteins, PrP 27-30 is a proteolytic cleavage prod-uct of a precursor protein, PrP 33-35d. However, PrP 33–35d is not theprimary product of the cellular gene. It has an amino acid sequenceand posttranslational modifications (like glycosylation and the attach-ment of GPI, glycophospholipid inositol anchor) identical to those of PrP33–35c, but strikingly different physicochemical features; in particular,PrPc is completely degraded by a limited proteolysis but PrPd is onlypartially degraded, yielding a core protein (PrP 27–30) which may bevisualised by electron microscopy as scrapie-associated fibrils (SAF),better known as prion rods13. To become PrPd, PrPc must be first trans-ported to the cell surface and then through the endosomal-lysosomalpathway.

PrP has several interesting features. As already mentioned, PrP isa glycoprotein with two Asn-glycosylation sites; thus, PrP may exist asdeglycosylated, monoglycosylated and di-glycosylated isoforms of dif-ferent electrophoretic mobilities and glycoforms14. The various combi-nations of glycosylation and codon 129 genotype (see later) correlate tosome degree with the phenotypic expression of human TSE. In partic-ular, a distinctive glycosylation pattern is uniquely present in both BSEand vCJD14,15. Although glycosylation patterns breed true—i.e., they areretained in passage14—changes in electrophoretic mobility may occurin the presence of metal ions14, and more than one pattern may occurin different regions of the same brain, or brain and peripheral organs inthe same patient.

PRNP gene in humans consists of two exons and the whole ORF isconfined to the second exon16. The polymorphism at codon 129 meritsspecial comment. Codon 129 encodes Met in ca 60% and Val in 40%of alleles in the normal Caucasian population. However, in all formsof CJD, there is marked over-representation of homozygotes over het-erozygotes. The codon 129 polymorphism may also exert a modifyingeffect on the phenotypic expression of a given PRNP mutation.

The situation in kuru is particularly interesting. The practice of can-nibalism underlying the kuru epidemic created a selective force onthe prion protein genotype. As in CJD, homozygosity at codon 129(129Met Met or 129Val Val) is overrepresented in kuru. However, Meadet al.17 found that among Fore women over fifty years of age, there isa remarkable overrepresentation of heterozygosity (129Met Val) at codon129, which is consistent with the interpretation that 129Vam Met makesan individual resistant to TSE agents and that such a resistance wasselected by cannibalistic rites. Because of this 129Met Val heterozygoteadvantage, it has been suggested that the heterozygous genotype atcodon 129 has been sustained by a widespread ancient practice ofhuman cannibalism.


2.4. Classifications

From early days, CJD (the name as Jakob-Creutzfeldt disease wascoined by Spielmeyer in 1922) has been sub-classified into severalforms. For instance, Daniel18 singled out the classical cortico-striato-spinal (Jakob) type; Heindenhain type (characterized by cortical blind-ness due to severe involvement of the occipital lobes); diffuse type(dementia with pyramidal and extrapyramidal signs and symptoms) andataxic type19. Siedler and Malamud20 discriminated cortical, cortico-striatal, cortico-striato-cerebellar, cortico-spinal and cortico-nigral type.In the literature, CJD exists under more than 50 different names andmany of these do not represent CJD in a modern sense. The dis-crimination of all these variants is merely of historical interest but re-cent molecular studies substantiated the existence of certain definedphenotypes.

PrPres (after limited proteolytic digestion) may exist as 21 kDa(type 1) and 19 kDa (type 2) isoforms which coupled with the statusof codon 129 of the PRNP gene underlie the existence of 7 molec-ular variants—MM1, MV1, MM2- cortical, MM2—thalamic, MV2, VV1and VV2. These variants differ both clinically and neuropathologically21.Type MM1corresponds to classical sporadic CJD with changes in thecerebral cortex, striatum, thalamus and the cerebellum; PrPd accumu-lates mostly as synaptic deposits. This type comprises approximately70% of sCJD cases. Second, most common type, VV2 comprises ap-proximately 15% of all sCJD cases. Changes are confined to the limbicsystem, striatum, the cerebellum, thalamus and hypothalamus and sev-eral brain stem nuclei. The involvement of the cerebral cortex dependson the duration of illness; those cases of short duration may exhibit mini-mal cortical changes, spongiform change demonstrates laminar distribu-tion while PrPd accumulates as plaque-like, perineuronal and synapticdeposits. MV2 type (approximately 8%) is reminiscent of VV2 type—spongiform change is confined to the subcortical structures while PrPd

expression is mostly plaque-like. In contrast to MV2 type, in VV2 type—“true” (i.e., congophilic and visible in a routine H & E stain) plaquespredominates. MM2 type is further sub-classified into MM2-thalamic,which corresponds to FFI and FSI cases and MM2-cortical, similar toMM1 type, from which differs by limited cerebellum involvement andlarger (coarse) vacuoles. VV1 is very rare (<1% of all sCJD)—changesare limited to cerebellar cortex and the striatum while other structures,including the cerebellum are barely involved.

A more refined approach was used by Collinge et al.14,22 who ex-ploited the size of PrPd fragments following limited PK digestion and therelative abundance of mono-, di- and deglycosylated glycoforms. This


approach discriminated PrPd types 1–4 and 6; type 5 exists in vCJD-infected transgenic mice but not in humans. All type 1 cases are homozy-gous for Met at codon 129 of the PRNP gene; type 2 may exist coupledwith every status of the codon 129; type 3 is associated with at leastone 129Val allele with the exception of a single CJD cases homozygousfor Met at codon 129. Type 4, characterized by predominance of digly-cosylated glycoform, is unique for vCJD and BSE15. These types differneuropathologically as well as clinically. Type 1 cases demonstratedwidespread spongiform change in the cerebral cortex, mild changes inthe basal ganglia, cerebellum and brainstem but no spongiform degener-ation in the hippocampus. In type 2 homozygous for 129Met cases, basalganglia are moderately affected while in heterozygous type 2 cases ortype 2 homozygous for 129Val, the basal ganglia are involved severely.Type 3 MV cases are characterized by the presence of kuru plaquesalready seen on routine H & E preparation.

The translation of the Collinge scheme into the Gambetti is notstraightforward, probably due to technical differences in the method-ology for Western blots. Furthermore, chelation of metal ions performedprion to PK digestion interconverts both type 1 and 2 MM PrP fragmentsinto so called 2− PrP23. Having said this, the Collinge’s type 1 MM,type 2 MM, type 3 VV, type 2 MV and type 3 MV are similar to theGambetti’s type MM1, MM2-cortical, VV2, MV1 and MV2, respectively.Thus, it seems that the Collinge sub-classification and the Gambetti sub-classification are, basically, interconvertible. This notion has been sup-ported by recent work which indicates that alterations in electrophoreticmobility can be markedly influenced by pH variations in the brain tissuehomogenate. When pH is controlled, it appears that two major sub-groups of PrPres can be identified in terms of electrophoretic mobilityof the unglycosylated band, corresponding to the types 1 and 2 of theGambetti et al. classification.

2.5. Classical Neuropathology

Creutzfeldt in 192024 described one case of a novel neurodegener-ative conditions and Jakob described sequentially 5 cases25,26. FourJakob’s cases, still on files at the University of Hamburg, were reexam-ined by Masters and Gajdusek27 who confirmed that 2 Jakob’s casesfulfill modern criteria of CJD while remaining 2 cases represent other notwell defined neurological conditions. Of special interest is one of Jakob’scases with amyotrophy which initiated a long-lasting confusion of “amy-otrophic type of CJD” that appear to be merely amyotrophic lateral scle-rosis with dementia and which is not transmissible28. Neuropathological


Figure 2.1. (a) Typical spongiform change. H & E; (b) status spongiosus; (c) Spongi-form vacuoles as seen by electron microscopy. Vacuoles contain curled membranefragments and secondary chambers. Original magnification, ×12 000; (d) Intramyelinvacuole, Original magnification, ×12 000.

description of Jakob’s cases based on studies of thick celloidin-embedded sections stained according to the Nissl technique re-vealed purely neurodegenerative process encompassing neuronal loss,central chromatolysis and astroglial proliferation with neuronophagia.Parenthetically, spongiform change were not visible by Nissl stain butre-appeared when the coverslips were removed and section re-stainedwith H & E.

The classical triad of CHD neuropathology consists of vacuolation(spongiform change), neuronal degeneration (neuronal loss) and astro-cytosis (Figure 2.1–2.2). The changes are bilaterally symmetrical butmay be local and, occasionally, even unilateral29.

2.6. Structural Changes

2.6.1. Spongiform changes

Most characteristic and even “semi-pathognomic” for CJD is thepresence of spongiform change which remain well preserved even


Figure 2.2. (a) Dense astrocytic gliosis in a CJD case as revealed by Cajal goldsublimate method. Courtesy of Prof. Herbert Budka, Vienna, Austria; (b) GFAP-immunopositive astrocytes against a background of severe spongiform change. CJDbrain biopsy.

in exhumed cases30. Spongiform change consists of small, round oroval vacuoles within neuropil (Figure 2.1); vacuoles are confluent andform typical “morula-like” aggregates. In the cerebral cortex, spongi-form change is confined to the deep cortical layers; those vacuoles inthe superficial cortical layers are characteristic for fronto-temporal lobardegenerations including Pick disease or are merely artefactual. It mustbe stressed, that in cases of longer duration spongiform change may bemasked by the overall loss of neurons, collapse of the cortical cytoar-chitecture and robust proliferation of astrocytes. To this end, Mastersand Richardson31 discriminated “spongiform change” from “spongiformstate (“status spongiosus”),’ the latter consisting of larger cavities of ir-regular shape in the neuropil (Figure 2.1b) between dense meshworkof proliferating astrocytes. Status spongiosus is not specific for TSEsand can occur in the end stage of a wide range of neurodegenerativedisorders if widespread neuronal degeneration and loss has occurred.

neurodegeneration and prion...

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