Proteasome Activity and Biological Properties of Normal Prion Protein: A Comparison between Young and Aged Cattle
Date: 18 Jul 2010 J-STAGE Advance Published Date: 2 Aug 2010
Proteasome Activity and Biological Properties of Normal Prion Protein: A Comparison between Young and Aged Cattle
Yumi YOSHIOKA1), Naotaka ISHIGURO1) and Yasuo INOSHIMA1)
1) Laboratory of Food and Environmental Hygiene, Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University
(Received 10-Apr-2010) (Accepted 18-Jul-2010)
ABSTRACT. Atypical bovine spongiform encephalopathy (atypical BSE) has recently been identified in several countries including Japan. Most cases of atypical BSE have been reported in cattle older than 8 years of age. To clarify the association between age and occurrence of atypical BSE, we investigated both the physiological properties and amount of cellular prion protein (PrPC) in brain homogenates from young and aged cattle by enzyme-linked immunosorbent assay and immunoblotting. The amount of PrPC in the brain homogenates was not significantly different between young and aged cattle, but the amount in the detergent-insoluble fraction in the aged cattle was significantly higher than that of young cattle. Significant differences were observed in neither of the glycosylation forms nor in proteinase K sensitivity in young and aged cattle. Age-related changes included deposition of lipofuscin pigment and a decrease of 33% in proteasome activity in the brains of aged cattle compared to that of young cattle.
KEY WORDS: atypical BSE, brain homogenate, cattle, prion, proteasome
To clarify the relationship between aged cattle and the occurrence of atypical BSE, we focused on the physiological properties and amount of PrPC in brain homogenates from young and aged cattle. In this study, the brains of aged cattle (> 120 months) exhibited a significant decrease in the 20S proteasome activity, and deposition of lipofuscin pigment served as a sign of aging. Oxidative metabolism is essential for neurons to generate energy, because the high energy demands in neurons lead to aging vulnerability . Additionally, lipofuscin contains some materials derived from lysosomal degradation; furthermore, it increases with aging .
We believe our study is support for the age-related decreases of protein turnover in cells. In our study, the amount of PrPC in the detergent-insoluble (P2) fraction was significantly higher in the young than in the aged cattle (Fig. 2C), which agrees well with a decrease in protein turnover including 20S proteasome activity with aging. In vitro evidence has shown that the 20S proteasome selectively degrades damaged proteins following an oxidative insult , and is a marker of the primary mechanism for the degrading oxidized proteins . Age-related decline in proteasome activity is also reported in the brain  and spinal cord .
To analyze more details of the physiological properties of PrPC, the S1 fraction was 10 then fractionated into detergent-soluble (S2) and detergent-insoluble (P2) proteins. PrPC amounts in the S2 and P2 fractions of the aged cattle was slightly higher than that of young cattle, while PrPC amount of the S1 fraction was almost same in young and aged cattle (Fig. 2A). Several experiments using proteasomal inbibitors such as MG132 and N-acetyl-leucinal-leucinal-norleucinal (ALLN) revealed that wild type PrPC is accumulated in both detergent-soluble and - insoluble species, when cells are incubated with proteasomal inhibitors [14, 22]. In particular, the insoluble fraction includes an unglycosylated 26 kDa PrPC molecules with a protease-resistant core [14, 22]. Thus in our study, the remarkable accumulation of PrPC in the P2 fraction of the aged cattle may be caused by the significant decreases in the 20S proteasome activity (Figs 1 and 2). In our study, the P2 samples from young and aged cattle were relatively resistant to PK treatement. However, it is not clear how the physiological properties of PrPC such as PK resistance and accumulation of PrPC in the P2 fraction of the aged cattle are associated with occurrence of atypical BSE. In the future, to elucidate the relationship between expression of PrPC and occurrence of atypical BSE, brain samples from atypical BSE need to be characterized directly.
We found influences of aging on neither of the glycosylation forms nor on PK sensitivity in the bovine PrPC. However, Goh et al. described both an increasing prevalence in the complex oligosaccharides in PrPC from aged mice and an absence of any relationship between aging and PK sensitivity of mouse PrPC . We conclude that age-related physiological changes in PrPC are not the same in cattle and mice. Likewise, Salès et al reported that PrPC expression increase with age in the brain of hamsters , while in the present study, the amount of PrPC in the S1 fractions were similar in young and aged cattle, suggesting that PrPC expression may not change 11 drastically with age in the brains of cattle. Again, we conclude interspecies differences.
The present study provides insight into possible explanations for the correlation between cattle age and occurrence of atypical BSE. Perhaps aging should not be considered strictly as an influence on PrPC, but also as an influence on variable structures and functions of the brain due through genetic and environmental mechanisms. Despite the plethora of research on neurodegenerative diseases associated with aging in humans, such as Alzheimer’s and Parkinson’s diseases, questions concerning aging still remain . Mitochondrial dysfunction is thought to be a key factor in age-related diseases of humans  and should therefore be investigated in the cattle. Biological relationships between aging and PrPC, including antioxidant-like activity , synaptic transmission , and the expression of PrPC exposure to oxidative stress in the aged mice , have also been reported. Thus, it is conceivable that PrPC plays a role in protecting neurons from the effects of cellular stress.
The origin and mechanisms of the occurrence atypical BSE remains unknown, as with classical BSE. Only one cases of atypical BSE have been reported in young cattle, while classical BSE occurred in aged cattle in Japan [13,21]. It is very difficult to precisely estimate the influences of age on atypical and classical BSE cases. Atypical BSE and classical BSE display unique incubation periods, PrPSc deposition patterns, and histological lesions [9,16]. However, because the onset of BSE is affected by exposure dose of BSE agent and host susceptibility, it is difficult to estimate the infection time for both classical and atypical BSE cases. There is a possibility that atypical BSE agents could infect young cattle and reside within the body for long periods before symptoms of BSE appears in aged cattle. In addition, the low prevalence of atypical BSE identified through mainly active surveillance means that whole brains are not often available for 12 examination. Thus the need remains for detailed sampling of the brain, with the 247 exception the obex region, to determine the etiology of atypical BSE.
HOW many cases were missed in the USA and all of North America ???
USDA: In 9,200 cases only one type of test could be used
WASHINGTON (AP)--The U.S. Department of Agriculture acknowledged Aug. 17 that its testing options for bovine spongiform encephalopathy were limited in 9,200 cases despite its effort to expand surveillance throughout the U.S. herd.
In those cases, only one type of test was used--one that failed to detect the disease in an infected Texas cow.
The department posted the information on its website because of an inquiry from The Associated Press.
Conducted over the past 14 months, the tests have not been included in the department's running tally of BSE tests since last summer. That total reached 439,126 on Aug. 17.
"There's no secret program," the department's chief veterinarian, John Clifford, said in an interview. "There has been no hiding, I can assure you of that."
Officials intended to report the tests later in an annual report, Clifford said.
These 9,200 cases were different because brain tissue samples were preserved with formalin, which makes them suitable for only one type of test--immunohistochemistry, or IHC.
In the Texas case, officials had declared the cow free of disease in November after an IHC test came back negative. The department's inspector general ordered an additional kind of test, which confirmed the animal was infected.
Veterinarians in remote locations have used the preservative on tissue to keep it from degrading on its way to the department's laboratory in Ames, Iowa. Officials this year asked veterinarians to stop using preservative and send fresh or chilled samples within 48 hours.
The department recently investigated a possible case of BSE that turned up in a preserved sample. Further testing ruled out the disease two weeks ago.
Scientists used two additional tests--rapid screening and Western blot--to help detect BSE in the country's second confirmed case, in a Texas cow in June. They used IHC and Western blot to confirm the first case, in a Washington state cow in December 2003.
"The IHC test is still an excellent test," Clifford said. "These are not simple tests, either."
Clifford pointed out that scientists reran the IHC several times and got conflicting results. That happened, too, with the Western blot test. Both tests are accepted by international animal health officials.
>>> These 9,200 cases were different because brain tissue samples were preserved with formalin, which makes them suitable for only one type of test--immunohistochemistry, or IHC. <<<
THIS WAS DONE FOR A REASON! THE IHC test has been proven to be the LEAST LIKELY to detect BSE/TSE in the bovine, and these were probably from the most high risk cattle pool, the ones the USDA et al, SHOULD have been testing. ...TSS
We have to be careful that we don't get so set in the way we do things that we forget to look for different emerging variations of disease. We've gotten away from collecting the whole brain in our systems. We're using the brain stem and we're looking in only one area. In Norway, they were doing a project and looking at cases of Scrapie, and they found this where they did not find lesions or PRP in the area of the obex. They found it in the cerebellum and the cerebrum. It's a good lesson for us. Ames had to go back and change the procedure for looking at Scrapie samples. In the USDA, we had routinely looked at all the sections of the brain, and then we got away from it. They've recently gone back. Dr. Keller: Tissues are routinely tested, based on which tissue provides an 'official' test result as recognized by APHIS. Dr. Detwiler: That's on the slaughter. But on the clinical cases, aren't they still asking for the brain? But even on the slaughter, they're looking only at the brainstem. We may be missing certain things if we confine ourselves to one area.
Dr. Detwiler: It seems a good idea, but I'm not aware of it. Another important thing to get across to the public is that the negatives do not guarantee absence of infectivity. The animal could be early in the disease and the incubation period. Even sample collection is so important. If you're not collecting the right area of the brain in sheep, or if collecting lymphoreticular tissue, and you don't get a good biopsy, you could miss the area with the PRP in it and come up with a negative test. There's a new, unusual form of Scrapie that's been detected in Norway. We have to be careful that we don't get so set in the way we do things that we forget to look for different emerging variations of disease. We've gotten away from collecting the whole brain in our systems. We're using the brain stem and we're looking in only one area. In Norway, they were doing a project and looking at cases of Scrapie, and they found this where they did not find lesions or PRP in the area of the obex. They found it in the cerebellum and the cerebrum. It's a good lesson for us. Ames had to go back and change the procedure for looking at Scrapie samples. In the USDA, we had routinely looked at all the sections of the brain, and then we got away from it. They've recently gone back.
Dr. Keller: Tissues are routinely tested, based on which tissue provides an 'official' test result as recognized by APHIS .
Dr. Detwiler: That's on the slaughter. But on the clinical cases, aren't they still asking for the brain? But even on the slaughter, they're looking only at the brainstem. We may be missing certain things if we confine ourselves to one area.
snip... FULL TEXT;
Completely Edited Version PRION ROUNDTABLE Accomplished this day, Wednesday, December 11, 2003, Denver, Colorado 2005
NOW, how many mad cows do you think were missed to the blatant deliberate carelessness of the testing and testing protocols, along with blatant flawed surveillance for BSE in the USA and all of North America $$$
CDC DR. PAUL BROWN TSE EXPERT COMMENTS 2006
The U.S. Department of Agriculture was quick to assure the public earlier this week that the third case of mad cow disease did not pose a risk to them, but what federal officials have not acknowledged is that this latest case indicates the deadly disease has been circulating in U.S. herds for at least a decade. The second case, which was detected last year in a Texas cow and which USDA officials were reluctant to verify, was approximately 12 years old. These two cases (the latest was detected in an Alabama cow) present a picture of the disease having been here for 10 years or so, since it is thought that cows usually contract the disease from contaminated feed they consume as calves. The concern is that humans can contract a fatal, incurable, brain-wasting illness from consuming beef products contaminated with the mad cow pathogen. "The fact the Texas cow showed up fairly clearly implied the existence of other undetected cases," Dr. Paul Brown, former medical director of the National Institutes of Health's Laboratory for Central Nervous System Studies and an expert on mad cow-like diseases, told United Press International.
"The question was, 'How many?' and we still can't answer that." Brown, who is preparing a scientific paper based on the latest two mad cow cases to estimate the maximum number of infected cows that occurred in the United States, said he has "absolutely no confidence in USDA tests before one year ago" because of the agency's reluctance to retest the Texas cow that initially tested positive. USDA officials finally retested the cow and confirmed it was infected seven months later, but only at the insistence of the agency's inspector general.
"Everything they did on the Texas cow makes everything USDA did before 2005 suspect," Brown said. ...
CDC - Bovine Spongiform Encephalopathy and Variant Creutzfeldt ...
Dr. Paul Brown is Senior Research Scientist in the Laboratory of Central Nervous System ... Address for correspondence: Paul Brown, Building 36, Room 4A-05, ...
In this context, a word is in order about the US testing program. After the discovery of the first (imported) cow in 2003, the magnitude of testing was much increased, reaching a level of >400,000 tests in 2005 (Figure 4). Neither of the 2 more recently indigenously infected older animals with nonspecific clinical features would have been detected without such testing, and neither would have been identified as atypical without confirmatory Western blots. Despite these facts, surveillance has now been decimated to 40,000 annual tests (USDA news release no. 0255.06, July 20, 2006) and invites the accusation that the United States will never know the true status of its involvement with BSE.
In short, a great deal of further work will need to be done before the phenotypic features and prevalence of atypical BSE are understood. More than a single strain may have been present from the beginning of the epidemic, but this possibility has been overlooked by virtue of the absence of widespread Western blot confirmatory testing of positive screening test results; or these new phenotypes may be found, at least in part, to result from infections at an older age by a typical BSE agent, rather than neonatal infections with new "strains" of BSE. Neither alternative has yet been investigated.
please see full text ;
Wednesday, July 28, 2010
re-Freedom of Information Act Project Number 3625-32000-086-05, Study of Atypical BSE UPDATE July 28, 2010
Wednesday, July 28, 2010 Atypical prion proteins and IBNC in cattle DEFRA project code SE1796 FOIA Final report
Wednesday, March 31, 2010
Atypical BSE in Cattle North America
*****URGENT NOTE HERE ABOUT OIE AND ATYPICAL BSE*****
To date the OIE/WAHO assumes that the human and animal health standards set out in the BSE chapter for classical BSE (C-Type) applies to all forms of BSE which include the H-type and L-type atypical forms. This assumption is scientifically not completely justified and accumulating evidence suggests that this may in fact not be the case. Molecular characterization and the spatial distribution pattern of histopathologic lesions and immunohistochemistry (IHC) signals are used to identify and characterize atypical BSE. Both the L-type and H-type atypical cases display significant differences in the conformation and spatial accumulation of the disease associated prion protein (PrPSc) in brains of afflicted cattle. Transmission studies in bovine transgenic and wild type mouse models support that the atypical BSE types might be unique strains because they have different incubation times and lesion profiles when compared to C-type BSE. When L-type BSE was inoculated into ovine transgenic mice and Syrian hamster the resulting molecular fingerprint had changed, either in the first or a subsequent passage, from L-type into C-type BSE. In addition, non-human primates are specifically susceptible for atypical BSE as demonstrated by an approximately 50% shortened incubation time for L-type BSE as compared to C-type. Considering the current scientific information available, it cannot be assumed that these different BSE types pose the same human health risks as C-type BSE or that these risks are mitigated by the same protective measures.
Tuesday, August 03, 2010
Variably protease-sensitive prionopathy: A new sporadic disease of the prion protein
Friday, November 30, 2007
CJD QUESTIONNAIRE USA CWRU AND CJD FOUNDATION