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Primary myopathy and accumulation of PRPSC-like molecules in peripheral tissues of transgenic mice expressing a prion protein insertional mutation
Oral Presentation:
p-J
Alzheimer’s
Disease und Related Disorders
S.51
(Prims)
NEUROFIBRILLARY TANGLES, NEURITIC PLAQUES, AND CEREBROVASCULAR LESIONS AT DEATH AMONG DEMENTED AND COGNITIVELY NORMAL JAPANESE-AMERICAN MEN
Oral Presentation: Disorders (Prions)
Helen Petrovitch, Kuakirti Med Ctr, Honolulu, HI; Sandra C Steinhorn. Pacific Health Rcwxzrch Institute, Honolulu, HI; George W Ross, Dept ~jj Veteran ‘\ Affair.s. Honolulu, HI; William R Markesbery, Damn G Davis, &iv of Kentucky, Lexington. KY: John Hardman. Univ of Hawaii, Honolulu, HI: Lenore _I Luunrr, Nutionul Instirute on Aging, Bethesda, MD; Lon R Whrte, Pacific Health Research Insritute. Honolulu, HI This report compares structural changea in brains of demented and cognitively normal decedents in a population-based study to determine the likelihood of clinical dementia during life given that an individual has neocortical neurotibrillary tangles (NFT), neocortical neuritic plaques (NP). and cerebrovascular lesions. Impact of combinations of these structural lesiona on dementia is also evaluated. Among 3437 Japanese-American men who had cognitive function testing (CASI) between 1Y91 and 1993 as a part of the Honolulu-Asia Aging Study examination, there were 222 decedents with complete autopsy data available for analysis. Clinical diagnosis of dementia was based on published research criteria. Neuropathologists blinded to clinical findings evaluated cerebrovascular lesions, NFT, and NP. By DSM-IIILR criteria, 79 were demented, and 68 were cognitively normal during life. Excluded from analysis were 76 who did not meet dementia criteria but scored below the median on the CASI. Presence of NP/NFT was defined as at least one neocottical lesion and presence of cerebravascular lesions as at least one lacune, infarct, or hemorrhage. Demented cases were significantly older, had lower mean educational levels, and lower medialrCAS1 scores than nondemented cases. Among 9 with none of the lesions 3 (33%) were demented, among 14 with NFI as the only lesion type 3 (21%) were demented, among 4 with NP as the only lesion type 1 (25%) was demented, and among 22 with cerebrovascular lesions as the only lesion type 7 (32%) were demented. Among groups with combined lesions 15 IS (44%) of 34 with NP + NFf were demented, 15 (68%) of 24 with Nfl + cerebrovascualar lesions were demented, and 3 (75%) of 4 with NP + cerebrovascular lesions were demented. There were 36 with all three lesions and 32(89%) of these cases were demented. Results indicate that the combination of either NP or NFI with cerebrovascular lesions greatly increases the likelihood of clinical dementia compared to groups with any single lesion type (p=O.O14). The group with all three lesion types wac also significantly more likely to have dementia compared to any of the groups with a combination of two lesion types (p=O.O03)
12581
@-AMYLOID PEPTIDE DOES NOT BIND IRON AFTER FIXATION BUT SENILE PLAQUES DO.
Senile plaques in the cerebral cortices of patients who have died from Al/heimer’5 disease have been shown to have iron concentrations as high ab I.OmM. The wurce of this iron is not known. While it is presumed that iron binds to P-amyloid, thir has not been confirmed. The present study investigates whether senile plaques in human cortex and @-amyloid deposits in rat cortex can bind iron after fixation. Hwnan tissur:inferior temporal cortex from four Alrheimer’s patient\ was fixed in 10% formalin, sectioned at 50p.m. and stored in phosphate buffer with I% sodium aride. Rat tissue: four adult Wiatar rats were anaesthetised with Halothane and their parietal cortices injected with l.Opl of 1.OmM rat P-amyloid,.,,,(CA Peptide) or human P-amyloid,~,,,(US Peptide). The rats were killed 24hr later with sodium pentobarbitol (2COmg/kg IP), perfused with 4% paraformaldehyde, and their brains post-fixed for 4hrs. Alternate serial sections (IOO~m) were immunolabelled to show the presence of B-amyloid. The remaining rat sections, along with human sections, were processed with a modified Perl’s Etain to reveal ferric iron deposits. Prior to procesairig. half of these ectiona were pre-incubated with ferric ammonium citrate (O.OlM) for Zhrs. In iron-incubated rat sections. P-amyloid remained unlabeled for iron despite intense neuronal labeling. By contrast, iron was detected in senile plaque cores of all human sections, while in iron-incubated sections, plaques as well as cores were intensely labeled. Thus, when fixed with pxaformaldehyde pure P-amyloid peptide does not bind appreciable quantities of iron whereas senile plaques that have been fixed with formalin do. We conclude that a ligand other than P-amyloid may bind iron in senile plaques. Furthermore, some of the iron previously reported in plaques may have been bound after fixation.
Alzheimer’s
Disease
and
Related
TRANSMISSION TO NON-HUMAN PRIMATES INDICATES THAT KURU AS WELL AS THE ATAXIC (VVZ) OR KURUPLAQUE (MV2) VARIANTS OF SPORADIC CREUTZFELDTJAKOB DISEASE ARE CAUSED BY THE SAME STRAIN OF PRIONS. Piero Parchi. Univ of Bolognu, Bolopw Italy; Paul Brown, Lab CNS Stud), NINDS. NIH, Bethesda, MD; Sabinn Cape//an’, Univ of Bolo~no, B&gnu ItulJ; Brmurdino Ghefti, Indianu Unrv Sch Medicine, Indianapolis, IN; Clarence .I Gibbs, Lab CNS Study, NINDS. NIH, Bethesda. MD: Pier&i L Gumbetti. Univ Ho.spifrrl.r/Cu.w Western Reserve Unir: Cleveland, OH
of
Five variants of sporadic Creutzfeldt-Jakob dtsease (sCJD) and the sporadic form of fatal familial insomnia are now recognized within the group of sporadic human prion diseases. In a study of 3Ml rubjects, we demonstrated that 3 variants are by far the most common and include about 95 % of subjects. 70% of them Thaw the classic CJD phenotype, PrPsC type I (molecular mass of 21 kDn), and at lealt one methionine allele at codon 129 of the priori protein gene (MM1 or MV 1 variant). whereas 25% display the ataxlc and kutwplaque variants, PIP type 2 (molecular mass of 19 kDa). and valine homozygosity or heterorygosity at codon 129, respectwely (VVZ and MV2 variants). The striking similarities between the phenotype of the VV2 or MV2 sCJD variants and that of kuru strongly suggest that there variant\ and kuru are linked to the same prion strain. In this study we tested this hypothesis by comparing incubation time, lesion profile, pattern of deposition and physicochemical properties of PrP”‘ in squirrel monkeys (n= 55) inoculated with brain extracts from MM1 (n= 25). VV2 all 3 genotypes at codon 129). (n=8) or MV2 (n= 3) aCJD, and kuru (II= 8, including lnocula from kuru and VV2 or MV2 patients produced a longer incubation time (kuru= 985 ? 170 p< 0.05; VV2= 1010 -t 85 p< O.OCKll;MV2 973 -c 40days) than the inocula from the MMI subjects (772 2 1 IS days). Similarly, PrPsC from the monkeys injected with kuru, VV2 and MV2 inocula displayed a significantly different glycoform ratio and showed a slightly faster gel migration than the PIP“ from the animals injected with MM1 inocula. Furthermore, the monkeys receiving kuru and VV2 or MV2 inocula shared the same lesion profile and type of spongiosis, which, however, were stgnificantly different from those of the animals inJected with MMI inocula. Finally, both kuru and VV2 or MV2 mocula produced in the monkeys focal granular deposits of PrP immunoreactivity, whereas the MMI inocula produced a fine, reticular pattern of staining. It is concluded that kuru and the MV2 or VV2 variants of sCJD are caused by the same strain of the agent, which has distinct biological properties from that causing the most common, MM 1, \CJD phenotype.
12601
PRIMARY MYOPATHY AND ACCUMULATION OF PRI”“-LIKE MOLECULES IN PERIPHERAL TISSUES OF TRANSGENIC MICE EXPRESSING A PRION PROTEIN INSERTIONAL MUTATION.
A nine-octapeptide insertional mutation in the prim protein (PrP) gene is associated with a form of CreutLfeldt-Jakob disease in humans. Transgenic mice that express the mourr PrP homologue of this mutation (PG14) under control of a PrP promoter &splay a progressive neurological dirordrr characterized by ataxm, apoptosis of cerebellar granule cells, deposition of PrP, and astrogliosis. In addition, beginning at birth the mice uccumulatr mutant PrP molecules in thex brains that display the major bmchemical hallmarks of PrPs’, the pathogenic iaoform of PrP (Chiesa et al. Newro?~ 21:133Y-1351 [1998]). We have now investigated PC14 PrP expre\+ion in fhe peripheral tissues of the Tg mice. We found highest levels of mutant PrP m the bram and spinal cord, internxxlmte levels in skeletal muscle, heart and testis, and low level? in kidney, lung. spleen. intestine and stomach; this expression pattern is similar to that of sndogenous PrP. To determine whether the mutant protein expressed in these t!Csuey was converted to a xrapie-like isoform, we rubjected homogenates to ultracentrifugation to teyt detergent Insolubility, or to digestion with proteinaae K (PK) to test protease resistance. We found that 30.80% of PC14 PrP exprwed m the peripheral tissues was detergent-insoluble, and that digestion with PK (0.5-l kg/ml for 30 min at 37°C) yielded a protease-resistant PrP 27-30 fragment. These two properties were not displayed by transgenically encoded wild-type moPrP expressed in the peripheral tissues of control mice (Tg[WT]). Histological analysis of skeletal muscle, the peripheral tissue with the highest level of PG14 PrP, revealed feature5 indicative of a progressive primary myopathy, including central nuclei, necrotic and regenerating fibers, and variable fiber six. No pathological abnormalities were found in the other tissue? examined. or in peripheral tissues of Tg(WT) mice. These results indicate that the PG14 mutation structurally alters the protein in a way that promotes conversion to a PrPsc-like state regardless of the tissue context. and suggest that accumulation of PrP” could have deleteriom effects on skeletal muscle cells a\ well as neurons.
p-J
Alzheimer’s
Disease und Related Disorders
S.51
(Prims)
NEUROFIBRILLARY TANGLES, NEURITIC PLAQUES, AND CEREBROVASCULAR LESIONS AT DEATH AMONG DEMENTED AND COGNITIVELY NORMAL JAPANESE-AMERICAN MEN
Oral Presentation: Disorders (Prions)
Helen Petrovitch, Kuakirti Med Ctr, Honolulu, HI; Sandra C Steinhorn. Pacific Health Rcwxzrch Institute, Honolulu, HI; George W Ross, Dept ~jj Veteran ‘\ Affair.s. Honolulu, HI; William R Markesbery, Damn G Davis, &iv of Kentucky, Lexington. KY: John Hardman. Univ of Hawaii, Honolulu, HI: Lenore _I Luunrr, Nutionul Instirute on Aging, Bethesda, MD; Lon R Whrte, Pacific Health Research Insritute. Honolulu, HI This report compares structural changea in brains of demented and cognitively normal decedents in a population-based study to determine the likelihood of clinical dementia during life given that an individual has neocortical neurotibrillary tangles (NFT), neocortical neuritic plaques (NP). and cerebrovascular lesions. Impact of combinations of these structural lesiona on dementia is also evaluated. Among 3437 Japanese-American men who had cognitive function testing (CASI) between 1Y91 and 1993 as a part of the Honolulu-Asia Aging Study examination, there were 222 decedents with complete autopsy data available for analysis. Clinical diagnosis of dementia was based on published research criteria. Neuropathologists blinded to clinical findings evaluated cerebrovascular lesions, NFT, and NP. By DSM-IIILR criteria, 79 were demented, and 68 were cognitively normal during life. Excluded from analysis were 76 who did not meet dementia criteria but scored below the median on the CASI. Presence of NP/NFT was defined as at least one neocottical lesion and presence of cerebravascular lesions as at least one lacune, infarct, or hemorrhage. Demented cases were significantly older, had lower mean educational levels, and lower medialrCAS1 scores than nondemented cases. Among 9 with none of the lesions 3 (33%) were demented, among 14 with NFI as the only lesion type 3 (21%) were demented, among 4 with NP as the only lesion type 1 (25%) was demented, and among 22 with cerebrovascular lesions as the only lesion type 7 (32%) were demented. Among groups with combined lesions 15 IS (44%) of 34 with NP + NFf were demented, 15 (68%) of 24 with Nfl + cerebrovascualar lesions were demented, and 3 (75%) of 4 with NP + cerebrovascular lesions were demented. There were 36 with all three lesions and 32(89%) of these cases were demented. Results indicate that the combination of either NP or NFI with cerebrovascular lesions greatly increases the likelihood of clinical dementia compared to groups with any single lesion type (p=O.O14). The group with all three lesion types wac also significantly more likely to have dementia compared to any of the groups with a combination of two lesion types (p=O.O03)
12581
@-AMYLOID PEPTIDE DOES NOT BIND IRON AFTER FIXATION BUT SENILE PLAQUES DO.
Senile plaques in the cerebral cortices of patients who have died from Al/heimer’5 disease have been shown to have iron concentrations as high ab I.OmM. The wurce of this iron is not known. While it is presumed that iron binds to P-amyloid, thir has not been confirmed. The present study investigates whether senile plaques in human cortex and @-amyloid deposits in rat cortex can bind iron after fixation. Hwnan tissur:inferior temporal cortex from four Alrheimer’s patient\ was fixed in 10% formalin, sectioned at 50p.m. and stored in phosphate buffer with I% sodium aride. Rat tissue: four adult Wiatar rats were anaesthetised with Halothane and their parietal cortices injected with l.Opl of 1.OmM rat P-amyloid,.,,,(CA Peptide) or human P-amyloid,~,,,(US Peptide). The rats were killed 24hr later with sodium pentobarbitol (2COmg/kg IP), perfused with 4% paraformaldehyde, and their brains post-fixed for 4hrs. Alternate serial sections (IOO~m) were immunolabelled to show the presence of B-amyloid. The remaining rat sections, along with human sections, were processed with a modified Perl’s Etain to reveal ferric iron deposits. Prior to procesairig. half of these ectiona were pre-incubated with ferric ammonium citrate (O.OlM) for Zhrs. In iron-incubated rat sections. P-amyloid remained unlabeled for iron despite intense neuronal labeling. By contrast, iron was detected in senile plaque cores of all human sections, while in iron-incubated sections, plaques as well as cores were intensely labeled. Thus, when fixed with pxaformaldehyde pure P-amyloid peptide does not bind appreciable quantities of iron whereas senile plaques that have been fixed with formalin do. We conclude that a ligand other than P-amyloid may bind iron in senile plaques. Furthermore, some of the iron previously reported in plaques may have been bound after fixation.
Alzheimer’s
Disease
and
Related
TRANSMISSION TO NON-HUMAN PRIMATES INDICATES THAT KURU AS WELL AS THE ATAXIC (VVZ) OR KURUPLAQUE (MV2) VARIANTS OF SPORADIC CREUTZFELDTJAKOB DISEASE ARE CAUSED BY THE SAME STRAIN OF PRIONS. Piero Parchi. Univ of Bolognu, Bolopw Italy; Paul Brown, Lab CNS Stud), NINDS. NIH, Bethesda, MD; Sabinn Cape//an’, Univ of Bolo~no, B&gnu ItulJ; Brmurdino Ghefti, Indianu Unrv Sch Medicine, Indianapolis, IN; Clarence .I Gibbs, Lab CNS Study, NINDS. NIH, Bethesda. MD: Pier&i L Gumbetti. Univ Ho.spifrrl.r/Cu.w Western Reserve Unir: Cleveland, OH
of
Five variants of sporadic Creutzfeldt-Jakob dtsease (sCJD) and the sporadic form of fatal familial insomnia are now recognized within the group of sporadic human prion diseases. In a study of 3Ml rubjects, we demonstrated that 3 variants are by far the most common and include about 95 % of subjects. 70% of them Thaw the classic CJD phenotype, PrPsC type I (molecular mass of 21 kDn), and at lealt one methionine allele at codon 129 of the priori protein gene (MM1 or MV 1 variant). whereas 25% display the ataxlc and kutwplaque variants, PIP type 2 (molecular mass of 19 kDa). and valine homozygosity or heterorygosity at codon 129, respectwely (VVZ and MV2 variants). The striking similarities between the phenotype of the VV2 or MV2 sCJD variants and that of kuru strongly suggest that there variant\ and kuru are linked to the same prion strain. In this study we tested this hypothesis by comparing incubation time, lesion profile, pattern of deposition and physicochemical properties of PrP”‘ in squirrel monkeys (n= 55) inoculated with brain extracts from MM1 (n= 25). VV2 all 3 genotypes at codon 129). (n=8) or MV2 (n= 3) aCJD, and kuru (II= 8, including lnocula from kuru and VV2 or MV2 patients produced a longer incubation time (kuru= 985 ? 170 p< 0.05; VV2= 1010 -t 85 p< O.OCKll;MV2 973 -c 40days) than the inocula from the MMI subjects (772 2 1 IS days). Similarly, PrPsC from the monkeys injected with kuru, VV2 and MV2 inocula displayed a significantly different glycoform ratio and showed a slightly faster gel migration than the PIP“ from the animals injected with MM1 inocula. Furthermore, the monkeys receiving kuru and VV2 or MV2 inocula shared the same lesion profile and type of spongiosis, which, however, were stgnificantly different from those of the animals inJected with MMI inocula. Finally, both kuru and VV2 or MV2 mocula produced in the monkeys focal granular deposits of PrP immunoreactivity, whereas the MMI inocula produced a fine, reticular pattern of staining. It is concluded that kuru and the MV2 or VV2 variants of sCJD are caused by the same strain of the agent, which has distinct biological properties from that causing the most common, MM 1, \CJD phenotype.
12601
PRIMARY MYOPATHY AND ACCUMULATION OF PRI”“-LIKE MOLECULES IN PERIPHERAL TISSUES OF TRANSGENIC MICE EXPRESSING A PRION PROTEIN INSERTIONAL MUTATION.
A nine-octapeptide insertional mutation in the prim protein (PrP) gene is associated with a form of CreutLfeldt-Jakob disease in humans. Transgenic mice that express the mourr PrP homologue of this mutation (PG14) under control of a PrP promoter &splay a progressive neurological dirordrr characterized by ataxm, apoptosis of cerebellar granule cells, deposition of PrP, and astrogliosis. In addition, beginning at birth the mice uccumulatr mutant PrP molecules in thex brains that display the major bmchemical hallmarks of PrPs’, the pathogenic iaoform of PrP (Chiesa et al. Newro?~ 21:133Y-1351 [1998]). We have now investigated PC14 PrP expre\+ion in fhe peripheral tissues of the Tg mice. We found highest levels of mutant PrP m the bram and spinal cord, internxxlmte levels in skeletal muscle, heart and testis, and low level? in kidney, lung. spleen. intestine and stomach; this expression pattern is similar to that of sndogenous PrP. To determine whether the mutant protein expressed in these t!Csuey was converted to a xrapie-like isoform, we rubjected homogenates to ultracentrifugation to teyt detergent Insolubility, or to digestion with proteinaae K (PK) to test protease resistance. We found that 30.80% of PC14 PrP exprwed m the peripheral tissues was detergent-insoluble, and that digestion with PK (0.5-l kg/ml for 30 min at 37°C) yielded a protease-resistant PrP 27-30 fragment. These two properties were not displayed by transgenically encoded wild-type moPrP expressed in the peripheral tissues of control mice (Tg[WT]). Histological analysis of skeletal muscle, the peripheral tissue with the highest level of PG14 PrP, revealed feature5 indicative of a progressive primary myopathy, including central nuclei, necrotic and regenerating fibers, and variable fiber six. No pathological abnormalities were found in the other tissue? examined. or in peripheral tissues of Tg(WT) mice. These results indicate that the PG14 mutation structurally alters the protein in a way that promotes conversion to a PrPsc-like state regardless of the tissue context. and suggest that accumulation of PrP” could have deleteriom effects on skeletal muscle cells a\ well as neurons.