Models of prion disease

Details Models of prion disease Models of prion disease

2001-06-28

2004-07-27

Shukla, Ram R. (Department: 1632)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S325000, C435S455000, C435S320100

Reexamination Certificate

active

06767712

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to systems for studying neurodegenerative disorders, and in particular to systems for the study of prion-associated disease.
BACKGROUND OF THE INVENTION
Prions are infectious pathogens that cause central nervous system spongiform encephalopathies in humans and animals. Prions are distinct from bacteria, viruses and viroids. The predominant hypothesis at present is that no nucleic acid component is necessary for infectivity of prion protein. Further, a prion which infects one species of animal (e.g., a human) will not readily infect another (e.g., a mouse).
A major step in the study of prions and the diseases that they cause was the discovery and purification of a protein designated prion protein (“PrP”) [Bolton et al.,
Science
218:1309-11 (1982); Prusiner et al.,
Biochemistry
21:6942-50 (1982); McKinley et al.,
Cell
35:57-62 (1983)]. Complete prion protein-encoding genes have since been cloned, sequenced and expressed in transgenic animals. PrP
C
is encoded by a single-copy host gene [Basler et al.,
Cell
46:417-28 (1986)] and is normally found at the outer surface of neurons. A leading hypothesis is that prion diseases result from conversion of PrP
C
into a modified form called PrP
Sc
.
It appears that PrP
Sc
is necessary for both the transmission and pathogenesis of the transmissible neurodegenerative diseases of animals and humans. See Prusiner, S. B., “Molecular biology of prion disease,”
Science
252:1515-1522 (1991). The most common prion diseases of animals are scrapie of sheep and goats, and bovine spongiform encephalopathy (BSE) of cattle [Wilesmith, J. and Wells,
Microbiol. Immunol
. 172:21-38 (1991)]. Four prion diseases of humans have been identified: (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI) [Gajdusek, D. C.,
Science
197:943-960 (1977); Medori et al.,
N. Engl. J. Med
. 326:444-449 (1992)]. The presentation of human prion diseases as sporadic, genetic and infectious illnesses initially posed a conundrum which has been explained by the cellular genetic origin of PrP.
Most CJD cases are sporadic, but about 10-15% are inherited as autosomal dominant disorders that are caused by mutations in the human PrP gene [Hsiao et al.,
Neurology
40:1820-1827 (1990); Goldfarb et al.,
Science
258:806-808 (1992); Kitamoto et al.,
Proc. R. Soc. Lond
. 343:391-398. latrogenic CJD has been caused by human growth hormone derived from cadaveric pituitaries as well as dura mater grafts [Brown et al.,
Lancet
340:24-27 (1992)]. Kuru, which for many decades devastated the Fore and neighboring tribes of the New Guinea highlands, is believed to have been spread by infection during ritualistic cannibalism [Alpers, M. P.,
Slow Transmissible Diseases of the Nervous System
, Vol. 1, S. B. Prusiner and W. J. Hadlow, eds. (New York: Academic Press), pp. 66-90 (1979)].
The initial transmission of CJD to experimental primates has a rich history beginning with William Hadlow's recognition of the similarity between kuru and scrapie. In 1959, Hadlow suggested that extracts prepared from patients who died of kuru be inoculated into nonhuman primates and that the animals be observed for disease that was predicted to occur after a prolonged incubation period [Hadlow, W. J.,
Lancet
2:289-290 (1959)]. Seven years later, Gajdusek, Gibbs and Alpers demonstrated the transmissibility of kuru to chimpanzees after incubation periods ranging form 18 to 21 months [Gajdusek et al.,
Nature
209:794-796 (1966)]. The similarity of the neuropathology of kuru with that of CJD [Klatzo et al.,
Lab Invest
. 8:799-847 (1959)] prompted similar experiments with chimpanzees and transmissions of disease were reported in 1968 [Gibbs, Jr. et al.,
Science
161:388-389 (1968)]. Over the last 25 years, about 300 cases of CJD, kuru and GSS have been transmitted to a variety of apes and monkeys.
The expense, scarcity and often perceived inhumanity of such animal experiments have restricted this work and thus limited the accumulation of knowledge. While the most reliable transmission data has been said to emanate from studies using nonhuman primates, some cases of human prion disease have been transmitted to rodents but apparently with less regularity [Gibbs, Jr. et al.,
Slow Transmissible Diseases of the Nervous System
, Vol. 2, S. B. Prusiner and W. J. Hadlow, eds. (New York: Academic Press), pp. 87-110 (1979); Tateishi et al.,
Prion Diseases of Humans and Animals
, Prusiner et al., eds. (London: Ellis Horwood), pp. 129-134 (1992)].
The importance of understanding the conversion of PrP
C
into PrP
Sc
has been heightened by the possibility that bovine prions have been transmitted to humans who developed variant Creutzfeldt-Jakob disease (vCJD), G. Chazot, et al.,
Lancet
347:1181 (1996); R. G. Will, et al.,
Lancet
347:921-925 (1996). Earlier studies had shown that the N-terminus of PrP
Sc
could be truncated without loss of scrapie infectivity, S. B. Prusiner, et al.,
Biochemistry
21:6942-6950 (1982); S. B. Prusiner, et al.,
Cell
38:127-134 (1984) and correspondingly, the truncation of the N-terminus of PrP
Sc
still allowed its conversion into PrP
Sc
(M. Rogers, et al.,
Proc. Natl. Acad. Sci. USA
90:3182-3186 (1993)).
Recent studies have advanced our ability to visualize the structural transition of PrP
c
to PrP
Sc
at a molecular level. For example, the N-terminal portion of PrP
C
is relatively unstructured and flexible, but assists in stabilizing structural elements within the C-terminal portion. D. G. Donne et al.,
Proc. Natl. Acad. Sci. USA
94:13452-13457 (1997). Furthermore, immunological studies have demonstrated that N-terminal epitopes are cryptic in PrP
Sc
, supporting the idea that this region undergoes profound conformational change during prion propagation. Peretz et al.,
J. Mol. Biol
. 273:614-622 (1997).
Despite these advances, our understanding of the structural biology of the pathogenic conversion process remains incomplete in many ways. For example, it is unknown exactly which structural regions of PrP
C
are necessary or sufficient for conformational change to occur. It is also unknown which regions of PrP
Sc
are necessary or sufficient for infectivity. Evidence indicates that prion strain phenomena and species barriers are a result of different PrP conformations, but the precise structural determinants of these conformations have not yet been precisely identified. Telling et al.
Science
274:2079-2082 (1996); Billeter, et al.,
Proc. Natl. Acad. Sci. USA
94:7281-7285 (1997).
Recent studies have identified four residues of mouse PrP (MoPrP) that appear to interact with protein X, a putative factor postulated to facilitate the conformational change from PrP
C
to PrP
Sc
. Telling, et. al.
Cell
83:79-90 (1995). All four amino acids come together to form the putative protein X binding site in the tertiary structure of recombinant PrP 90-231 and PrP 29-231. D. G. Donne et al.,
Proc. Natl. Acad. Sci. USA
94:13452-13457 (1997); T. L. James et al.,
Proc. Natl. Acad. Sci. USA
94:10086-10091 (1997). However, despite several reports of proteins which bind PrP
C
, the identity of protein X remains elusive. Finally, although the structures of refolded, recombinant PrP molecules may resemble PrP
C
, a structural solution for PrP
Sc
remains lacking.
One method of studying prion disease and the physiological changes inherent in the disease is to alter the physical structure of the PrP
C
protein expressed in infected cells to examine the effect on progression of prion-mediated disorders. In particular, the glycosylation sites of PrP
C
were initially examined to determine their role in conversion of PrP
C
to PrP
Sc
. PrP
C
mutants with Thr to Ala substitutions in two NXT consensus glycosylation sites (182 and 198) exhibited increased sporadic conversion to a proteinase K-resistant form following transfecti

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