Prion protein hereditary amyloidosis: parenchymal and vascular

seminars in V I R O L O G Y , Vol 7, 1996: pp 189–200 Prion protein hereditary amyloidosis: parenchymal and vascular Bernardino Ghetti, Pedro Piccardo, Blas Frangione*, Orso Bugiani†, Giorgio Giaccone†, KatherineYoung, Frances Prelli*, Martin R. Farlow, Stephen R. Dlouhy and Fabrizio Tagliavini† th e n ewly recogn ized PrP cerebral amyloid an giopath y ( PrP-CAA) . GSS h as been well characterized clin ically, path ologically, bioch emically, an d genetically, wh ile PrP-CAA is still n ot well known. In GSS, the amyloidosis is paren ch ymal wh ile in PrP-CAA it is predomin an tly vascular. Prion protein (PrP) amyloidosis is a feature of Gerstmann-Sträussler-Scheinker disease (GSS) and prion protein cerebral amyloid angiopathy (PrP-CAA). GSS and PrP-CAA are associated with point mutations of the prion protein gene (PRNP); there is a broad spectrum of clinical presentations and the main signs are ataxia, spastic paraparesis, extrapyramidal signs and dementia. In GSS, parenchymal amyloid may be associated with spongiform changes or neurofibrillary lesions; in PrP-CAA, vascular amyloid is associated with neurofibrillary lesions. In the two diseases, a major component of the amyloid fibrils is a 7 kDa peptide, approximately spanning residues 81–150 of PrP. The ‘H’ family and the development of the concept of Gerstmann-Sträussler-Scheinker disease In 1912, 1928 an d 1936, two patien ts with a n eurological disorder th at had affected multiple gen eration s of th eir family ( family ‘H’) were described clin ically an d path ologically.1-3 Th ey presen ted with cerebellar ataxia, dysdiadochokinesia, an d action and in ten tion tremor. Later, th ey were unable to walk, stan d or even sit upright and had disturban ces of speech , swallowin g difficulties, lateral and vertical gaze n ystagmus, decreased lower extremity reflexes, an d bilateral Babin ski sign. Ch an ges in personality, specifically irritability, un con trolled beh avior, labile mood, an d decreased intellectual per forman ce also appeared. Subsequen tly, addition al members of th is family presen ted symptoms similar to those of the first two patien ts reported.4-8 Neuropath ologically, Gerstman n et al 3 described atroph y of th e cerebral h emispheres and the cerebellar vermis, an d deposits of amorph ous material in th e cerebellar an d cerebral cortices, basal gan glia, an d wh ite matter th at were reminiscent of sen ile plaques of Alzh eimer disease ( AD) ; Alzh eimer neurofibrillary degen eration was n ot present. Since the deposits h ad th e tin ctorial properties of amyloid, Seitelberger 6 emph asized th eir similarity to kuru plaques. A reexamin ation of brain tissue of patients of th e ‘H’ family revealed also a mild to moderate spon giosis. Con sisten t with th is observation is th e fact th at recen tly, in a patien t of the ‘H’ family, the disease h ad a relatively sh ort course an d its man ifestation s Key words: amyloid / cerebrovascular amyloidosis / Gerstmann-Str äussler-Sch ein ker disease / prion protein / PRNP gene ©1996 Academic Press Ltd T H E TERM PRIO N PRO TEIN ( PrP) amyloidosis is used to describe a group of diseases in wh ich th e accumulation of PrP degradation products occurs in th e form of multiple distin ct fibrillary deposits. In PrP amyloidosis, diffuse n on fibrillary PrP deposits are also foun d in association with th e amyloid. Accumulation in brain paren ch yma of abn ormal PrP isoforms with out a sign ifican t deposition of fibrillary PrP occurs in the subacute spon giform en ceph alopath ies, ( sporadic, familial, an d iatrogen ic Creutzfeldt-Jakob disease {CJD}) , an d fatal familial in somn ia; h owever, in kuru, th e cen tral n ervous system ( CNS) , particularly th e cerebellum, may con tain deposits of PrP-amyloid ( kuru plaques) . Typically PrP-amyloid is seen in Gerstman n -Str äussler-Sch ein ker disease ( GSS) an d in From the Departments of Pathology and Laboratory Medicine, Medical and Molecular Genetics, and Neurology, Indiana University School of Medicine, Indianapolis, IN 46202-5120, USA, *Department of Pathology, New York University Medical Center, New York, NY 10016, USA and †Istituto Neurologico Carlo Besta, 20133 Milano, Italy ©1996 Academic Press Ltd 1044-5773/ 96/ 030189 + 12 $18.00/ 0 189 B. Ghetti et al were clin ically an d path ologically similar to th ose of CJD. Masters et al,9 wh o reexamin ed th e clin icopath ological data related to several familial syn dromes similar to th at in th e ‘H’ family, called th e con dition Gerstman n -Str äussler syn drome an d reported th at wh ile amyloid deposits were a con stan t feature, spon giosis was n ot an d th at in oculation of brain tissue from some patien ts caused a spon giform en ceph alopath y in th e recipien t an imal. Th us, a relation sh ip between GSS an d th e tran smissible spon giform en ceph alopath ies, such as CJD, was foun d. In 1986, amyloid of GSS was immun olabeled usin g an tibodies again st PrP10 an d in 1989, th e first mutation causin g GSS was discovered in th e PrP gen e ( PRNP) at codon 102.11 Th is mutation was also foun d in patien ts of th e ‘H’ family.12 foun d in AD an d oth er disorders are presen t in th e n eocortex an d subcortical n uclei. Thus, differen t gen etic types of GSS may be delineated on the basis of clin ical an d n europath ological presen tation. The clin ical ph en otypes are associated with mutation s of PRNP, allelic polymorph ism, and possibly with environ men tal an d tissue-specific factors. PrP cerebral amyloid angiopathy ( PrP-CAA) is a n ewly recogn ized ph en otype characterized by demen tia associated with a stop mutation at codon 145 of PRNP an d deposition of PrP amyloid in cerebral vessels in con jun ction with abun dan t n eurofibrillary lesion s.17 PRNP genotypes and clinicopathological phenotypes: GSS and PrP-CAA Codon 102 mutation (P102L) Gerstmann-Sträussler-Scheinker disease and the definition of parenchymal and vascular prion protein amyloidosis A prolin e ( CCG) to leucin e ( CTG) substitution on a Met129 allele h as been found in at least 32 families from th e USA, Can ada, the United Kin gdom, German y, Fran ce, Austria, Italy, Israel and Japan 1,8,11,16 an d is th e most common cause of GSS. Th e clin ical ph en otype is ch aracterized by a slowly progressive cerebellar syn drome with ataxia, dysarth ria an d in coordin ation of saccadic movemen ts. Pyramidal an d pseudobulbar signs are often seen . Men tal an d beh avioural deterioration leadin g to mild demen tia or akin etic mutism occur in th e late stages of disease. Th e age at on set of clinical sign s is in the fourth to sixth decades of life an d the duration of the disease ran ges from five mon ths to six years. Some families sh ow con siderable ph enotypic variability.8,15,16,18 Myoclon us an d pseudoperiodic sh arp wave disch arges ( PSD) in th e electroen cephalogram ( EEG) , a fin din g of diagn ostic relevan ce in CJD, occur on ly in a few P102L patien ts an d some of them presen t a rapid course of five to nine mon th s with a clin ical picture in distin guish able from that of CJD.19 Th e n europath ologic phen otype is ch aracterized by th e presen ce of deposits of fibrillar an d non-fibrillar PrP in th e cerebellar and th e cerebral paren chyma again st a backgroun d of variable spon giform changes ( Figures 1A–D) .8,9,18,20 Spon giform chan ge, neuron al loss an d astrocytosis vary in severity even among patien ts of th e same kin dred, and are most severe wh en th e course of th e illness is rapid.8, 18, 19 Recen tly, th e P102L mutation was detected in couplin g with a glutamin e to lysin e substitution at residue 219. Symptomatic subjects of that family had Five mutation s of th e PRNP gen e are curren tly kn own to be associated with GSS an d on e with PrP-CAA. In addition , polymorph isms at PRNP codon 129 ( Met or Val) an d 219 ( Glu or Lys) may play a role in th e path ogen esis of GSS.13,14 In fact, in th e patien ts of families studied to date, each specific GSS-causin g poin t mutation is in couplin g with th e same 129 codon .15 GSS is autosomal domin an t an d is ch aracterized clin ically by motor abn ormalities an d in tellectual deterioration , an d path ologically by PrP-amyloid deposits an d, in some cases, protein ase-K resistan t PrP in th e CNS. Symptoms occur in th e th ird to seven th decades; th e mean duration of illn ess is five years. Th e in ciden ce of GSS, believed to be less th an two per h un dred million , is probably un derestimated sin ce GSS may presen t as a syn drome mimickin g spin ocerebellar degen eration , olivopon tocerebellar atroph y, spastic paraparesis an d demen tia.16 Th e n europath ologic diagn osis of GSS is based on th e presen ce of PrP-amyloid deposits, th e distribution an d exten t of wh ich differ widely between families. Amyloid is accompan ied by glial proliferation an d by loss of n euron al processes an d perikarya, leadin g to variable degrees of atroph y of th e affected region s. Spon giform degen eration is n ot con sisten tly foun d. In some families, n umerous n eurofibrillary lesion s ( n eurofibrillary tan gles ( NFT) , n europil th reads, abn ormal n eurites) in distin guish able from th ose 190 Parenchymal and vascular PrP amyloidosis dred,29,35 severe ataxia, dysarth ria, mild Parkin sonism an d demen tia in a patient from a th ird family.30 In th e Alsatian an d th e US–German families, th e age at the on set of th e clin ical signs is 19–64 years an d 23–58, respectively,33,35 wh ile th e duration of th e disease ran ges from on e to 11 and two to six years, respectively. Th e clin ical ph en otype in the Alsatian family is variable;34 alth ough th e three patients studied in th e first an d secon d gen erations of the published pedigree exh ibited demen tia as the main clin ical symptom, affected subjects from subsequen t generation s sh owed th e association of dementia, pyramidal an d pseudobulbar sign s with ataxia, extrapyramidal symptoms, amyotroph y, myoclon us an d ton ic–clon ic seizures. EEG did n ot sh ow PSD. Th e n europath ologic phen otype is ch aracterized by presen ce of PrP-amyloid deposits th at are widespread th rough out th e cerebrum, but rare or absent in the cerebellum of subjects with demen tia alone.30,31,33-35 Numerous PrP-amyloid deposits in the cerebral cortex, basal gan glia an d th alamus as well as in the cerebellum were foun d in th ree patien ts from the Alsatian family,34 th at h ad died at 24, 39 and 73 years. In th e latter, wh o survived n in e years after the on set of th e clin ical sign s, NFT were n umerous in th e cerebral cortex, in con trast with th e rare deposits of β-amyloid ( Aβ) . Spon giform ch an ges are variable in severity and exten t. In patien ts of the US-German family, PrPamyloid deposits are prominen t in th e cerebral cortex an d striatum, but n ot in th e cerebellum; NTF are occasion ally foun d. In th e recen tly reported third family, th ere is a con spicuous deposition of PrP amyloid in th e cerebellum.30 demen tia but n ot ataxia. Neuropath ologic studies demon strated mild PrP deposition an d n o spon giform ch an ges.14 Tran sgen ic mice expressin g h igh levels of th e mutan t P101L PrP ( amin o acid 101 in mouse PrP correspon ds to amin o acid 102 in h uman PrP) spon tan eously develop a n eurologic illn ess ch aracterized by ataxia, leth argy, bradykin esia an d rigidity. Neuropath ologically, PrP deposits in cerebrum an d cerebellum an d vacuolar degen eration in th e n eocortex an d h ippocampus are observed.21 Codon 105 mutation (P105L) A prolin e ( CCA) to leucin e ( CTA) substitution at codon 105 on a Vall29 allele h as been foun d in patien ts with h ereditary spastic paraparesis from four Japan ese families.22-27 Th e clin ical ph en otype is ch aracterized by spastic gait, h yperreflexia an d Babin ski sign .26,27 Extrapyramidal sign s such as fin e fin ger tremor an d rigidity of limbs may be seen . Paraparesis progresses to tetraparesis an d is accompan ied by emotion al in con tin en ce an d demen tia. Myoclon us, PSD in EEG or severe cerebellar sign s h ave n ot been reported. Th e age at th e on set of th e clin ical sign s is in the fourth an d fifth decades of life, th e duration of th e disease ran ges from six to 12 years. Th e n europath ologic ph en otype is ch aracterized by presen ce of PrP deposits in th e n eocortex, especially th e motor area, striatum an d th alamus. In th e n eocortex, multicen tric PrP-amyloid an d diffuse deposits are presen t in super ficial an d deep layers respectively in association with n euron al loss an d astrocytosis. Neurofibrillary ch an ges are occasion ally seen , but n ot spon giform ch an ges. Amyloid plaques are rare in th e cerebellum, wh ile myelin loss occurs in th e pyramidal tracts.23-27 Codon 198 mutation (F198S): the Indiana kindred A ph en ylalan in e ( TTC) to serin e ( TCC) substitution at codon 198 on a Vall29 allele h as been described in patien ts from an In dian a kin dred.13,16,36,37 Th e clin ical ph en otype is ch aracterized by a gradual loss of sh ort-term memory and progressive clumsin ess in walkin g, bradykin esia, rigidity, dysarthria, an d demen tia. Sign s of cogn itive impairmen t an d eyemovemen t abn ormalities may be detected by specific tests before clin ical on set of symptoms. Psych otic depression h as been seen in several patien ts; tremor is mild or absen t. Symptoms may progress slowly over five years or rapidly over as little as on e year. Th e age at on set of clin ical sign s is 40–71 years; patien ts h omozygous for Val at codon 129 have clinical sign s Codon 117 mutation (A117V) An alan in e ( GCA or GCG) to valin e ( GTG) substitution on a Vall29 allele h as been described in a Fren ch ( Alsatian ) family an d two US kin dreds, on e bein g of German origin .28-20 It is possible th at th e Alsatian an d US–German families sh are a common foun der because th e ch an ge from GCA to GTG th at th ey sh are would require two mutation s, an d GCG is rare in th e n ormal population . Th e clin ical ph en otypes are presen ile demen tia in th e Alsatian family,31-34 presen ile demen tia, pyramidal sign s, an d parkin son ism in th e US–German kin 191 B. Ghetti et al n erve cell loss an d gliosis in th e n eocortex, striatum, red n ucleus, substan tia nigra, cerebellum, locus coeruleus, an d in ferior olivary n ucleus, and iron deposition in th e globus pallidus, striatum, red n ucleus an d substan tia n igra are found. Spon giform ch an ges are in con spicuous. A n eurologically asymptomatic subject, wh o had th e F198S mutation an d was h eterozygous at codon 129, died at th e age of 42. In this subject, PrP-amyloid deposits were already n umerous in th e cerebellum but rare in th e cerebrum. more th an 10 years earlier, on average, th an h eterozygous patien ts.13 Th e duration of th e disease ran ges from two to 12 years.16,37,38 Th e n europath ologic ph en otype is ch aracterized by presen ce of PrP-amyloid deposits in th e cerebral paren ch yma as well as n eurofibrillary lesion s in th e cerebral gray matter ( Figures 1E–G) .16,37,39-41 In th e 10 patien ts an d th e on e n on -symptomatic carrier studied, un icen tric an d multicen tric PrP-amyloid deposits, ran gin g between 10 an d 160 µm, are distributed th rough out th e gray structures of th e cerebrum, cerebellum an d midbrain . Amyloid deposition is severe in frontal, in sular, temporal an d parietal cortex; th e h igh est con cen tration of deposits is in layers on e, four, five an d six ( Figures 1E–F) . A moderate in volvemen t is seen in th e h ippocampus wh ere plaques occur predomin an tly with in th e stratum lacun osum-moleculare of th e CA1 sector an d subiculum. PrP deposits are n umerous in th e claustrum, th e caudate n ucleus, putamen , th e an terior, dorsomedial, ven trolateral an d lateral dorsal n uclei of th e th alamus, th e cerebellar molecular layer, th e mesen ceph alic tegmen tum, th e substan tia n igra, an d periaqueductal grey matter. Amyloid deposits are surroun ded by astrocytes, astrocytic processes an d microglial cells. In th e n eocortex, man y amyloid cores are associated with abn ormal n eurites, so th at wh en th ese lesion s are an alysed with classical stain s, th ey are morph ologically similar to n euritic plaques of AD.37,39 Th e n eurites immun oreact with an tibodies to τ ( Figure 1G) , ubiquitin , an d to N- an d C-termin al domain s of th e β-amyloid precursor protein ( βPP) . Th e accumulation of βPP in n erve cell processes is n ot associated with extracellular deposition of Aβ-protein , except in older patien ts wh ere Aβ immun oreactivity may also be observed aroun d PrP-amyloid deposits.16,42 Neurofibrillary tan gles are foun d in large n umber in th e n eocortex ( Figure 1G) an d in th e subcortical grey matter. Cortical region s particularly affected are th e fron tal, cin gulate, parietal, in sular an d parah ippocampal cortex. In th e latter, th e n euron s of th e secon d an d fifth layers are most in volved. In th e remain in g cortical region s, NFT are presen t but less n umerous. In most cases, NFT are n ot foun d in th e CA1 th rough CA3 areas of th e h ippocampus, but may be presen t in CA4. Of th e subcortical grey areas, th e n ucleus basalis, th e midbrain an d pon tin e n uclei, th e substan tia n igra, th e griseum cen trale, an d th e locus coeruleus, sh ow a variable degree of in volvemen t. Neuropil th reads are mostly foun d in th e n eocortex. Moderate to severe cerebral an d cerebellar atroph y, Codon 217 mutation (Q217R) A glutamin e ( CAG) to argin in e ( CGG) substitution 36 on a Val129 allele h as been described in two patien ts from an American family of Swedish origin . Th e clin ical ph en otype is ch aracterized by gradual memory loss, progressive ataxia, Parkinson ism an d demen tia. Th e age at on set of clin ical sign s is 62–66 years. Th e duration of th e disease is five to six years.43 Th e n eurological sign s may be preceded by episodes of man ia or depression that respon d to an ti-depressan t medication s, lith ium and neuroleptics. Th e n europath ologic phen otype is ch aracterized by presen ce of PrP-amyloid deposits in the cerebrum and cerebellum an d abun dan t NFT in the cerebral cortex an d several subcortical gray structures. The n eocortex, amygdala, substantia inn omin ata an d th alamus are severely in volved. PrP and Aβ immun oreactivity are con sisten tly associated.44 Codon 145 mutation (Y145STOP) A tyrosin e ( TAT) to stop codon ( TAG) substitution on a Met129 allele 17 h as been found, through studies of brain autopsy tissue of a Japan ese patien t with a clin ical diagn osis of AD.45 Th e clin ical ph en otype is characterized by memory disturban ce, disorien tation , and a progressive, severe demen tia. Th e EEG did not sh ow PSD. Th e age at the on set of th e clin ical signs is 38 years, while th e duration of th e disease is 21 years. Th e n europath ologic phen otype is ch aracterized by presen ce of PrP-amyloid deposits in the wall of small an d medium-sized parench ymal an d leptomenin geal blood vessels ( Figures 2A–C) an d in the perivascular n europil as well as n eurofibrillary lesion s in the cerebral gray matter ( Figures 2D–E) . Diffuse atroph y of th e cerebrum, dilation of the lateral ven tricles, n euron al loss an d gliosis are severe; th ere are n o spon giform ch an ges. 192 Parenchymal and vascular PrP amyloidosis Figure 1. ( A–D) Gerstman n -Str äussler-Scheinker disease with mutation P102L. ( A) Uni- and multicen tric amyloid deposits in th e cerebral cortex; Thioflavin S stain, 3 136. ( B) Multiple PrP deposits in th e cerebral cortex; immunostaining with monoclon al an tibody 3F4 following hydrolytic autoclavin g, 3 138. ( C) Amyloid deposits ( arrows) in the cerebral cortex; Hematoxylin and Eosin stain s, 3 140. ( D) Amyloid deposits ( arrows) and spongiform ch an ges in th e cerebral cortex; Hematoxylin an d Eosin stain s, 3 140. ( E–G) Gerstman n-Str äussler-Sch einker disease with mutation F198S. ( E) Un i- an d multicen tric amyloid deposits an d n eurofibrillary tangles in th e cerebral cortex; Th ioflavin S stain , 3 154. ( F) Multiple PrP deposits in the cerebral cortex; immun ostain in g with an tiserum to synthetic peptide homologous to the midregion of PrP, ( residues 90-102) , 3 98. ( G) Neurofibrillary lesions in nerve cell bodies and cell processes in th e cerebral cortex; immun ostain in g with monoclon al antibody PHF1, 3 96. 193 B. Ghetti et al ph en otype in th e former is ch aracterized by PrPamyloid deposits an d spongiform changes, wh ile in th e latter it is ch aracterized by PrP-amyloid deposits an d NFT in th e absen ce of spon giform ch an ges. In PrP-CAA, th e amyloid, in volvin g blood vessels and th e adjacen t paren ch yma, coexists with NFT in the absen ce of spon giform ch an ges. Paren ch ymal PrP-amyloid deposits are birefrin gen t after Con go red stain, stron gly fluorescent when observed un der ultraviolet ligh t after thioflavine S Th e clin ical an d path ologic ph en otypes of h ereditary PrP amyloidosis are summarized in Table 1. PrP-Amyloid Th e spectrum of lesion s in PrP-amyloidosis varies accordin g to th e PRNP gen otype. In GSS, th e spectrum is wide an d th e mutation s P102L an d F198S represen t th e two extremes; th e n europath ologic Figure 2. ( A–E) Prion protein cerebral amyloid angiopathy. ( A) Vascular amyloidosis ( arrowh ead) and n eurofibrillary tan gles ( arrow) in the cerebral cortex; Th ioflavin S stain , 3 150. ( B) Paren ch ymal vessels of th e cerebral cortex cut in th e longitudin al and tran sversal plan es show PrP deposits; immun ostain in g with an tiserum to synth etic peptide h omologous to th e midregion of PrP, ( residues 90-102) , 3 253. ( C) Vessels of the cerebellar cortex cut in th e lon gitudin al an d transversal plan es sh ow fluorescen t amyloid deposits; Th ioflavin S stain, 3 229. ( D) Neurofibrillary tan gles in th e h ippocampus; Thioflavin S stain , 3 153. ( E) Neurofibrillary lesion s in n erve cell bodies an d cell processes in th e hippocampus; immunostain ing with mon oclon al an tibody PHF1, 3 162. 194 Parenchymal and vascular PrP amyloidosis n europil. Th ese data suggest th at amyloid deposition in GSS is accompan ied by accumulation of PrP peptides oth er th an th e amyloid protein.41 Non -fibrillar PrP appears as diffuse punctate immun ostain in g of th e n europil an d is best eviden ced using h ydrolytic autoclavin g of brain section s before PrP immun oh istoch emistry. In th e P102L ph en otype, two types of PrP deposits are presen t. Some deposits h ave an amyloid core best recogn ized by an tibodies directed to epitopes in th e PrP region 90–165 an d a n on -amyloid periphery labelled by an tisera to th e N- an d C-termini. Oth er deposits are immun olabeled with equal in tensity th rough out by an tibodies raised against th e midregion as well as th e C- an d N-termin i of PrP.20 Bioch emical studies have been carried out in F198S, Q217R an d A117V. Protein sequencing of peptides extracted from amyloid cores isolated from th e brain of patien ts with F198S sh ows th at a major compon en t of amyloid fibrils is an 11 kDa peptide span n in g residues 50–150 of PrP.46 Further studies reveal th at th e smallest amyloid subunit is a 7 kDa peptide exten din g from residues 81 to 150 in F198S, from 81 to 146 in Q217R, an d from 81 to 146 in A117V47,48 ( Figures 3 and 4) . Thus, in three GSS varian ts, th e amyloid peptides correspond to intern al stain in g ( Figure 1C, 2A, 3A, 3C) , an d, at th e electron microscopic observation , appear to be composed of bun dles of fibrils radiatin g out from a cen tral core, each fibril measurin g 8–10 n m in diameter. In th e F198S varian t, immun oh istoch emical studies usin g mon oclon al an tibody 3F4 ( recogn izin g an epitope at PrP residues 109–112) an d an tisera to syn th etic peptides h omologous to th e N-termin al ( residues 23–40) , th e repetitive region ( residues 58–71) , th e mid-region ( residues 90–102, 127–147, an d 151–165) , an d C-termin al ( residues 220–231) domain s of h uman PrP sh ow th at th e cerebral an d cerebellar amyloid deposits with an d with out a n euritic compon en t are immun oreactive with an tisera to th e mid-region , wh ile th ey are un reactive or on ly weakly labeled by an tisera to th e N- an d C-termin i epitopes. Th is suggests th at th e amyloid protein is an in tern al fragmen t of PrP. On th e oth er h an d, an tisera to th e N- an d C-termin i of PrP label th e periph ery of amyloid cores as well as large areas of th e n europil th at do n ot possess th e tin ctorial an d optical properties of amyloid. Immun ogold electron microscopy sh ows th at an tibodies to th e mid-region of PrP decorate fibrils of amyloid cores wh ile an tisera to Nan d C-termin al epitopes label amorph ous material at th e periph ery of th e cores or dispersed in th e 195 B. Ghetti et al fragmen ts of PrP, a fin din g con firmed by immun oh istoch emistry. In addition to 11 kDa an d 7 kDa peptides, amyloid fraction s purified from patien ts with F198S or Q217R varian ts also con tain larger PrP fragmen ts.46,47 Th is is con sisten t with th e immun oh istoch emical observation th at th e n europil surroun din g amyloid deposits con tain s n on -fibrillary PrP material labeled by an tisera to N- an d C-termin al domain s of PrP. Th e amyloid peptides in F198S an d Q217R varian ts do n ot in clude th e region con tain in g th e amin o acid Figure 4. Schematic represen tation of the prion protein and of the amyloidogen ic fragments. substitution , wh ile th e amyloid peptide in A117V does. In GSS, th e amyloid peptides appear to origin ate on ly from th e mutan t allele, sin ce in patien ts with F198S an d Q217R, an d h eterozygous at codon 129, on ly Val was foun d at position 129 an d in patien ts with A117V, on ly mutan t Val 117 was present in th e amyloid subun it protein.47,48 Bioch emical studies h ave been carried out in GSS P102L patien ts. Western blot an alysis with antibodies to PrP sh ow th at th e amyloid-en rich ed fraction con tain ed 25–30 an d 15–20 kDa bands as well as a lower molecular weigh t peptide. N-terminal sequencin g reveals th at th e N-termin us of th e 25–30 and 15–20 kDa peptides corresponds to the octapeptide repeat region , alth ough determin ation of its exact localization was h ampered by peptide h eterogeneity. Amin o acid sequen ce analysis of peptides obtain ed by en zymatic digestion of th e 25–30 and 15–20 kDa protein s sh ows th at th e mutant leucin e at position 102 is con tain ed in th e amyloid fraction.49,50 To in vestigate th e PrP sequen ces th at are important for amyloid fibril formation in GSS, syn th etic peptides h omologous to con secutive segmen ts of the 11 kDa an d 7 kDa amyloid peptides purified from F198S or Q217R patien ts were used for in -vitro fibrillogen esis.51 Th ese studies sh ow that peptides correspon din g to residues 106–126 an d 127–147 of PrP readily form Figure 3. Partial purification of GSS amyloid proteins by gel filtration chromatograph y an d immun oblot an alysis of th e major amyloid protein fraction with an ti-PrP antibodies ( inset) . Proteins were extracted by formic acid from amyloid cores and fraction ated on a Seph adex G-100 column. Protein peaks were collected an d pooled as indicated ( fractions 1–7) . Gel filtration yielded two major peaks: the void volume ( fraction 1) an d a low molecular weight peak ( fraction 6) that was presen t as a broad ban d centered at ~ 7 kDa on SDS-polyacrylamide min igels. Th is band was immunoreactive with an tisera to syn th etic peptides h omologous to PrP residues 90–102 ( lan e b) an d 127–147 ( lan e d) as well as with th e mon oclon al an tibody 3F4 to residues 109–112 ( lane c) . Con versely, it was un reactive with antisera to PrP residues 23–40 ( lan e a) an d 200–231 ( lane e) , indicating that th e amyloid protein was an in ternal fragment of PrP. 196 Parenchymal and vascular PrP amyloidosis filamen ts measures about 22–24 n m at its maximum width , an d th e h elical twist has a period of about 70–80 n m. NFT h ave been observed in some GSS patien ts with P105L an d A117V varian ts. Immun ocytoch emical studies in F198S with antibodies Alz50, Tau-46 and Tau-1, recogn izin g epitopes at th e N- an d C-termin i an d in th e mid-region of th e τ protein , respectively, show immun opositivity of n euron s with NFT. Tau-1 requires dephosph orylation before immun ostain in g, sin ce th e epitope recogn ized by th is an tibody is ph osph orylation depen den t. The NFT may also be stain ed with an tibodies against ubiquitin . In addition to the NFT, such an tibodies also reveal th e n euritic componen t, surroun din g th e PrP amyloid deposits of th e neocortex, as well as the n europil th reads. Th us, th e cytoskeletal alteration s observed in n erve cell bodies an d neurites in GSS are in distin guish able from those observed in AD. By immun ogold electron microscopy, Alz50 an d Tau46 recogn ize th e intracellular paired h elical filamen ts.57 Paired h elical filamen t-en rich ed fraction s obtained from th e n eocortex of F198S patien ts con tained SDSsoluble τ isoforms with electrophoretic mobility and immun och emical profile correspon din g to those of A68 extracted from the brain of Alzheimer patien ts. In fact, th ese protein s migrate between 60 an d 68 kDa, immun oreact with an tibodies to the N- and C-termin i of τ, an d require dephosph orylation to be accessible to Tau-1. Th us, th e immun ocytoch emical fin din gs are con sisten t with th ose of the Western blot an alysis sh owin g th at n o differen ce exists between F198S GSS an d AD as to th e Alz50, T46 an d Tau-1 immun ostain in g of NFT. In PrP-CAA, NFT, n europil th reads an d dystroph ic n eurites are n umerous in th e cerebral gray matter an d are labelled by an tibody ALZ50, recognizing an epitope in residues 2–10 of τ an d by phosph orylationdepen den t an ti-τ an tibody AT270, AT8, AT180, an d PHF1, recogn izin g ph osph orylated Thr181, Ser202– Th r205, Th r231, Ser396–Ser404, respectively. Abn ormal n eurites immun olabeled by ALZ50, AT8 an d PHF1 are closely associated with paren ch ymal amyloid deposits an d amyloid laden blood vessels, resultin g in a picture of n euritic immunostain in g that overlaps with PrP imun oreactivity.17 fibrils th at possess th e tin ctorial properties an d X-ray diffraction pattern s of n ative amyloid. It is n oteworth y th at th e syn th etic peptide span n in g residues 106–126 is toxic on n euron s an d troph ic on astroglial cells in culture.52,53 In GSS, several molecules are associated with PrP in th e amyloid deposits. Th ese in clude P-compon en t, complemen t factors, apolipoprotein -E, apolipoprotein -J an d h eparan sulfate.46, 54-56 Such molecules, wh ose role in th e path ogen esis of amyloidosis is still un determin ed, are associated with various types of amyloid origin atin g from diverse protein s. Furth ermore, in th e GSS varian ts kn own to date, PrP deposits may be accompan ied by Aβ deposition . Th is association is gen erally observed in elderly GSS patien ts. Such colocalization may suggest th at th e two protein s or th eir fragmen ts h ave biological or ch emico-ph ysical relation sh ips. In PrP-CAA, fibrils are seen adjacen t to an d with in th e vessel wall. PrP-amyloid deposits are immun olabeled with an tibodies to PrP span n in g th e region 90–147 an d n ot with an tibodies to Aβ. PrP is n ot reactive with PrP23–40, wh ile labelin g occurs with an tiserum to the C-termin us, suggestin g th at n ormal PrP or C-termin al fragmen ts co-exist with vascular amyloid.17 By immun oblot an alysis of protein s extracted from purified amyloid fraction s, it appears th at th e smallest amyloid subun it migrates as a broad ban d cen tered at 7.5 kDa. Th is ban d is stron gly immun oreactive with an tibodies to epitopes located with in residues 90–147 of PrP, an d is un reactive with an tisera to N- an d C-termin i. In addition , amyloid fraction s con tain h igh er molecular weigh t PrP peptides migratin g as poorly resolved ban ds of 12–16 an d 22–30 kDa. Th ese ban ds exh ibit th e same pattern of immun oreactivity as th e 7.5 kDa peptide, suggestin g th at th ey con sist primarily of polymers of amyloid protein . Neurofibrillary tangles NFT in cell bodies an d in n eurites are a major feature of th e n europath ologic ph en otype in GSS varian ts F198S an d Q217R as well as PrP-CAA ( Y145STOP) ( Figures 2C, 3D–E) .37,39,40,43,57 NFT h ave th e tin ctorial properties of amyloid, i.e. birefrin gency after Con go red an d fluorescen ce un der ultraviolet ligh t after th ioflavin e S stain in g. By electron microscopy, paired h elical filamen ts appear as th e main con stituen ts of th e NFT. Each member of th e pair is a filamen t about 10 n m in diameter. Th e pair of Animal transmission studies Masters sh owed th at th e in oculation of brain h omogen ates from th ree patients affected by GSS into n on 197 B. Ghetti et al D. Nochlin, D. Schiffer an d H.V. Vinters for providing tissue of GSS patien ts, Dr Marco R. Celio for providing th e antibody against calcium binding protein, Dr Peter Davies for providing the an tibody Alz-50, and Dr Ivan Lieberburg for providing the antibody 10D5. h uman primates in some in stan ces in duced a spon giform en ceph alopath y in th e recipien t an imals.9 Th e patien ts were members of two Japan ese an d on e British family. Subsequen tly, in tracerebral in oculation in to marmosets from an oth er patien t of th e British family in duced a spon giform en ceph alopath y in distin guish able from th at seen in marmosets in oculated with brain tissue from a case of CJD.58 Tran smission experimen ts from P102L GSS patien ts to mice resulted in th e developmen t of spon giform degen eration .58, 59 It is sign ifican t th at th e don or tissue is ch aracterized by th e presen ce of PrP amyloid an d severe spon giform ch an ges, whereas th e recipien t primates an d roden ts develop a rapidly progressin g disease with severe spon giform degen eration but n ot PrP amyloid deposition . Tran smission of a spon giform en ceph alopath y is n ot con sisten tly obtain ed from P102L patien ts. Tran smission experimen ts, usin g A117V brain tissue h omogen ate, h ave been attempted several times usin g roden ts as recipien ts. Such studies h ave been n egative so far.60 In th e case of F198S, brain tissue an d buffy coat from on e affected in dividual were in oculated in to h amsters in two experimen ts in th e laboratory of Drs E. an d L. Man uelidis. No path ologic ch an ges were observed in th e primary tran smission attempt, n or in th e secon d an d th ird serial passage ( Dr L. Man uelidis, person al commun ication ) . Tissue h omogen ates from an oth er F198S an d on e A217G patien t h ave been in oculated in to h amsters an d mice an d n o tran smission h as occurred more th an 450 days after in oculation .36,37 Recen tly, amyloid en rich ed fraction s an d tissue h omogen ates from F198S patien ts h ave been in oculated in to marmosets an d in to tran sgen ic mice contain in g a n ormal h uman PRNP gen e ( Baker an d Collin ge, person al commun ication s) . 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