1976 — 1978 |
Prusiner, Stanley |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Biochemistry of Scrapie @ University of California-San Francisco |
0.915 |
1978 — 1981 |
Prusiner, Stanley |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Biochemical Studies of Scrapie in Cell Culture @ University of California-San Francisco |
0.915 |
1985 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Multiple Sclerosis and Viral Infections of the Cns - Sup @ University of California San Francisco |
0.936 |
1985 — 1990 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Viral Degenerative and Dementing Diseases in Aging @ University of California San Francisco
The occurrence of degenerative diseases increases with aging. Degeneration of the central nervous system (CNS) leaves patients devastated with presenile or senile forms of dementia. While the etiologies of most forms of dementia remain obscure, the number of demented patients is progressively increasing as the proportion of our population comprised of older people continues to grow. Toward gaining a better understanding of these age-dependent degenerative changes, we plan to focus our research on viral degenerations of the CNS. The dramatic degeneration of the CNS produced by the scrapie and Creutzfeldt-Jakob Disease (CJD) slow transmissible agents make them excellent models for studying degenerative diseases. In this program application we propose purification and characterization studies of the scrapie agent, studies on the biological and biochemical properties of the CJD agent, development of tissue culture systems for propagation of the CJD agent and studies of the pathogenesis and ultrastructural morphology of experimental CJD. Our research efforts will be of significance in the diagnosis, cure and possibly treatment of older patients with dementia as well as in defining the unusual properties of a novel class of microorganisms, having incubation periods measured in decades and capable of causing profound degeneration of the CNS.
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0.936 |
1985 — 1991 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Multiple Sclerosis and Viral Infections of the Cns @ University of California San Francisco
Considerable progress in understanding the structure and biology of prions as well as the diseases they cause has been made during the past year. Molecular cloning studies have elucidated the genetic origin and structure of the prion protein (PrP). This approach also led to the discovery of the cellular prion protein (PrPC). Ultrastructural studies have shown that prions may exist in at least 3 different states: 1) membrane-bound form, 2) rods, and 3) spheres. Studies on the genetics of scrapie and CJD have shown that at least 2 host genes influence the incubation period. The second gene designated PDI-2 is linked to the prion protein gene. Scrapie is rapidly becoming the most well understood degenerative neurologic disorder. Studies on myelin basic protein (MBP) genes have led to chromosomal assignments in mice and humans, elucidation of the gene structure in mice and sequencing of the first exon from the human MBP gene. During the coming year, studies on the genetics and ultrastructure of scrapie prions will continue. Work on MBP gene structure, therapy and polymorphisms will be performed.
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0.936 |
1985 — 1988 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Sen Jacob Javits Center of Excellence in Neuroscience @ University of California San Francisco
The proposed Jacob Javits Center will be devoted to studies of the slow infectious agents or prions causing scrapie and Creutzfeldt-Jakob disease (CJD). Seven established, senior investigators will comprise the nucleus of this research effort. All the investigators have already participated in collaborative studies on prions and are continuing to do so. Recent, methodologic advances, including the production of antibodies to the scrapie prion protein and the cloning of the cDNA encoding this protein, provide a firm foundation for any new approaches to the study of degenerative neurologic diseases. Our investigations will focus on the structure of the prion, its mode of replication and the mechanisms by which it causes CNS degeneration. Additional studies are directed toward understanding the formation of prion amyloid plaques, the chemical pathology of scrapie and CJD, the structural differences between scrapie and CJD prions, identification of genes controlling the resistance and susceptibility of mice to scrapie, and determination of the role of prion genes in Gerstmann-Straussler syndrome and possibly Alzheimer's disease. The Javits Center provides an ideal vehicle for bringing together a group of talented scientists with expertise in a large number of highly specialized disciplines. The complexities of prion research program demand a multidisciplinary approach and require expertise in virology, immunology, molecular cloning, experimental neurology, protein chemistry, x-ray crystallography, neuropathology, cell biology, ultrastructure, physical biochemistry, murine genetics, amino acid sequencing, neuropeptide analysis, protein biosynthesis and processing, as well as protein structure analysis. All of the investigators have outstanding records of achievement in one or more of these scientific disciplines. Recent collaborations among these investigators have led to 1) molecular cloning and identification of host genes encoding the prion protein, 2) demonstration that amyloid plaques in the brains of animals infected with scrapie are composed of prion proteins, 3) identification of CJD prions isolated from human brains, and 4) compelling evidence demonstrating that prions are novel infectious pathogens and are not tyical viruses. Creation of a Javits Center will allow us to build upon these significant achievements. Learning about the novel structure and replication mechanisms of prions will undoubtedly lead to important revisions in our thinking about the pathogenesis of degenerative CNS disorders.
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0.936 |
1985 — 1991 |
Prusiner, Stanley B |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Degenerative Neurological Diseases @ University of California San Francisco |
0.936 |
1986 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Viral Degenerative Dementing Disease in Aging (Supp) @ University of California San Francisco |
0.936 |
1987 |
Prusiner, Stanley B |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Experimental Approaches to Degenerative Neurological Dis @ University of California San Francisco |
0.936 |
1989 — 1990 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Jacob Javists Center of Excellence in Neuroscience @ University of California San Francisco
Scrapie is a neurodegenerative disease that can be transmitted to experimental animals. The infectious scrapie agent exhibits many unusual properties which distinguish it from viruses and thus the name "prion" has been suggested to emphasize these differences. Convergence of many lines of experimental evidence asserts that the scrapie isoform of the prion protein (PrPSc) is a necessary component of the infectious scrapie prion. Six senior investigators with outstanding records of accomplishment in a variety of scientific disciplines and with demonstrated commitments to scrapie research propose to extend their collaborative studies currently supported by an NINDS-Senator Jacob Javits Center for Excellence in Neuroscience. The exciting progress in scrapie research over the past five years coupled with the complementary talents and skills of this group of scientists represent a unique opportunity to make further advances. As described in this application, we want to take advantage of some unprecedented discoveries in prion diseases of animals and humans. Molecular genetic studies in mice and humans have shown that susceptibility to prion diseases - experimental scrapie in mice and Gerstmann-Straussler syndrome (GSS) in humans - is genetically linked to mutations in the open reading frame of the prion protein (PrP) gene. Transgenic (Tg) mice expressing hamster (Ha) PrP genes exhibit susceptibility incubation times and amyloid plaques characteristic of hamsters. Even more significant, these Tg (HaPrP) mice produce HaPrPSc and Ha prions after inoculation with Ha prions. These results have created a transformation in prion research from an area formerly restricted to descriptive and correlative observations to one now amenable to experimental manipulation. We propose to determine if Tg mice harboring a GSS mutant PrP gene will develop scrapie spontaneously. We shall also use Tg mice to identify scrapie domains of HaPrP which are required for synthesis of Ha prion infectivity. Other studies will focus on the nature of scrapie "strains" as well as a continuing search for a putative scrapie-specific nucleic acid. Learning whether or not mutant PrP molecules are converted into PrPGSS and deposited as amyloid will be important in deciphering the pathogenesis of prion disorders. Recent results suggest that investigations of crystalline arrays of PrPSc may begin to yield useful structural information. Our studies may ultimately elucidate the mechanisms responsible for a variety of CNS degenerative disorders, in particular those of unknown etiology afflicting older people such as Alzheimer's disease.
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0.936 |
1989 |
Prusiner, Stanley B |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
Electron Microscope-Jeol 100cx @ University of California San Francisco
Electron microscopy has made important contributions to the study of scrapie over the past decade. Scrapie is a transmissible, neurodegenerative disease of sheep and goats. The scrapie agent is neither a virus nor a viroid and the term "prion" has been suggested for this class of novel infectious pathogens. Initially, the electron microscope was used to identify components of partially purified fractions of scrapie prions and define the ultrastructure of the particles found in these fractions. Rod- shaped particles were found in some fractions with high prion titers. Ultrastructural analyses of purified rods led to the discovery that scrapie prion proteins can form amyloid filaments which coalesce to amyloid plaques. Antisera raised against the scrapie prion protein, the only identifiable component of the prion was used to demonstrate the immunoreactivity of the prion rods and to identify extracellular collections of amyloid filaments composed of prion proteins in the brains of scrapie-infected hamsters. Ultrastructural analyses showed that the rods were formed as a consequence of detergent extraction of the microsomal membranes. The nature of the scrapie prion was further revealed by studies in which Purified rods were transformed into detergent-lipid-protein complexes (DLPC) by a mixture of cholate and phosphatidylcholine. Dialysis of the DLPC to remove detergent resulted in the formation of closed liposomes. Throughout the transformations from rods to DLPC to liposomes and back to rods, high levels of prion infectivity were maintained. No filamentous or rod-shaped particles were found amongst prion liposomes by electron microscopy. We anticipate that electron microscopy will continue to be an important technique in studies focused on: 1) prion structure, 2) the cell biology of prion proteins, and 3) pathogenic mechanisms of scrapie and CJD. The history of important discoveries in prion research emanating from ultrastructural studies provides excellent support for our view that electron microscopy will continue to be a necessary and important technique for future investigations. Because the scrapie and CJD agents are extremely difficult to inactivate and the CJD agent is a human pathogen, we were forced to set up our own electron microscopy suite 10 years ago. Because our JEOL 100B electron microscope was built in 1970, its useful lifetime is approaching an end. The instrument contains numerous vacuum tubes and other parts which are no longer manufactured. We no seek funds to replace our 18 year old electron microscope with a comprobable, new instrument.
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0.936 |
1990 — 1993 |
Prusiner, Stanley B |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Leadership and Excellence in Alzheimer's Disease @ University of California San Francisco
Alzheimer's disease (AD) is a rapidly growing problem of epidemic proportions. The cause of AD is unknown. The lack of a "perfect" animal model for AD has slowed progress in deciphering its etiology. While experimental scrapie in animals differs in some respects from AD, many aspects of the CNS degeneration caused by scrapie prions resembles that found in AD. We propose to exploit some recent, unprecedented discoveries in prion diseases of animals and humans. Molecular genetic studies in mice and humans have shown that susceptibility to prion diseases - experimental scrapie in mice and Gerstmann-Straussler syndrome (GSS) in humans - is genetically linked to mutations in the open reading frame (ORF) of the prion protein (PrP) gene. We plan to sequence the ORF's of PrP genes from patients with GSS and familial Creutzfeldt-Jakob disease (FCJD) defining the extent of human PrP gene variation. Additional linkage studies with new PrP gene mutations in families with GSS of FCJD will be undertaken. Sequencing of entire PrP genes from normal humans, patients of GSS, inbred mice with long and short incubation times, sheep breeds with different susceptibilities to scrapie and several species of hamsters with distinct incubation times is planned. A search for prion diseases involving proteins other than PrP is proposed. Grey tremor mice will be examined first since it appears to be both genetic and infectious like GSS. We plan to create physical maps for mice and humans extending about 5 Mb on each side of the PrP gene. YAC clones will be recovered and used to construct contig maps. Probes derived from these YAC clones will be employed to identify a linked scrapie incubation time gene (Prn-i) in mice and the GSS gene in humans if these prove to be separable from the PrP gene. We plan to identify PrP related genes in mammals and in lower organisms especially those amenable to genetic manipulation. As more is learned about prion diseases it may be possible to determine whether natural scrapie is a genetic or infectious disease - both mechanisms have been proposed. We plan a set of genetic linkage studies in sheep with scrapie. Random mutagenesis of PrP genes from rodents will be performed and expressed clones assessed for production of PrPSc and infectivity. Such studies may ultimately elucidate the mechanisms underlying diseases which are both infectious and genetic. Indeed, unprecedented discoveries in studies on prion diseases signal new directions in CNS degenerative disease research;the implications for Alzheimer's disease and some other neurodegenerative disorders of older people may be profound.
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0.936 |
1991 — 1993 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Degenerative &Dementing Diseases of Aging @ University of California San Francisco
Dementing and degenerative neurological deseases are responsible for many decastating human illnesses. As medical science has become progressively more successful, more people are living to an older age. With this change in demography has come an epidemic of a previously insignificant disorder-senile dementia. Most cases of senile dementia are due to Alzheimer's disease (AD). Not only is the cause of AD unknown, but there is no specific diagnostic test, and there is no effective treatment. The possible causes of AD include: a slow virus-like agent, a genetic disorder, accumulation of a toxin, or production of an abnormal metabolite. The many similarities between Creutzfeldt-Jakob disease (CJD) and AD have raised the possibility that a slow infectious agent might be the cause of AD; however, all attempts to transmit AD to laboratory animals have been disappointing. Recent progress has shown that a protein is required for the infectivity of the agent causing CJD and a similar neuodegenerative disease of sheep and goats, scrapie. Because the dominant molecular characteristics of these slow agents are those of a protein, and they are different from both viruses and viroids, we have chosen to label them "prions" Understanding the molecular structure of slow infectious pathogens such as prions, which cause both scrapie and CJD, may help us to understand the etiology of not only AD, but also other devastating degenerative diseases of unknown etiology which afflict primarily older people. Improved purification protocols led to the identification of a 27-30 kDs protein that is a component of the scrapie prion. The unique molecular properties of the scrapie agent, or prion, appear to have defined a new class of small infectious pathogens. In this proposal, we outline studies designed to elucidate 1) the complete chemical structures of the cellular and scrapie prion proteins, 2) the molecular mechanisms of scrapie pathogenesis, 3) the cellular metabolism of the scrapie and cellular prion proteins, and 4) structure of scrapie and CJD may become the most well understood neurodegenerative diseases.
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0.936 |
1991 — 1994 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Transmissible and Genetic Neurodegenerative Diseases @ University of California San Francisco
Scrapie is a neurodegenerative disease that can be transmitted to experimental animals. The infectious scrapie agent exhibits many unusual properties which distinguish it from viruses and thus the name "prion" has been suggested to emphasize these differences. Convergence of many lines of experimental evidence asserts that the scrapie isoform of the prion protein (PrPSc) is a necessary component of the infectious scrapie prion. Six senior investigators with outstanding records of accomplishment in a variety of scientific disciplines and with demonstrated commitments to scrapie research propose to extend their collaborative studies currently supported by an NINDS-Senator Jacob Javits Center for Excellence in Neuroscience. The exciting progress in scrapie research over the past five years coupled with the complementary talents and skills of this group of scientists represent a unique opportunity to make further advances. As described in this application, we want to take advantage of some unprecedented discoveries in prion diseases of animals and humans. Molecular genetic studies in mice and humans have shown that susceptibility to prion diseases - experimental scrapie in mice and Gerstmann-Straussler syndrome (GSS) in humans - is genetically linked to mutations in the open reading frame of the prion protein (PrP) gene. Transgenic (Tg) mice expressing hamster (Ha) PrP genes exhibit susceptibility incubation times and amyloid plaques characteristic of hamsters. Even more significant, these Tg (HaPrP) mice produce HaPrPSc and Ha prions after inoculation with Ha prions. These results have created a transformation in prion research from an area formerly restricted to descriptive and correlative observations to one now amenable to experimental manipulation. We propose to determine if Tg mice harboring a GSS mutant PrP gene will develop scrapie spontaneously. We shall also use Tg mice to identify scrapie domains of HaPrP which are required for synthesis of Ha prion infectivity. Other studies will focus on the nature of scrapie "strains" as well as a continuing search for a putative scrapie-specific nucleic acid. Learning whether or not mutant PrP molecules are converted into PrPGSS and deposited as amyloid will be important in deciphering the pathogenesis of prion disorders. Recent results suggest that investigations of crystalline arrays of PrPSc may begin to yield useful structural information. Our studies may ultimately elucidate the mechanisms responsible for a variety of CNS degenerative disorders, in particular those of unknown etiology afflicting older people such as Alzheimer's disease.
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0.936 |
1992 — 2002 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Cns Degeneration Caused by Novel Pathogens @ University of California San Francisco
The prion diseases like Alzheimer's disease (AD) are age-dependent, degenerative neurological disorders. CNS degeneration caused by prions is the most well understood of the disorders of delayed onset and they are the focus of this proposal. Much evidence argues that prion diseases are disorders of protein conformation. Investigation directed toward elucidating the molecular mechanism of conversion of the cellular prion protein) (PrP/c) into the scrapie isoform (PrP/sc) are proposed. Using molecular genetic approaches, we plan to extend recent findings showing that an artificial mutation (Ala>Val) in the first putative region of secondary structure in PrP produces neurodegeneration in transgenic (Tg) mice was found to adopt spontaneously a conformation which is high in beta-sheet and similar to that of PrP/sc. Additionally, we found that two large segments of PrP can be deleted with the resulting protein of 106 residues being converted into a soluble PrP/sc-like molecule. We propose to create additional amino acid substitutions and deletion constructs. We shall determine the NMR structures of soluble, full length wild-type (wt) and mutant PrPs that have been expressed in E. coli (Project 2). In parallel, we plan to determine the structures of the recombinant PrPs expressed both in E. coli and Tg mice by X-ray crystallography (Project 3). A third approach to determining the structures of PrP/c and PrP/sc utilizes batteries of recombinant antibodies (Project 4). Such antibodies have been produced and used to identify portions of the PrP molecule that refold during conversion of PrP/c into PrP/sc. The diverse skills, talents and backgrounds of the investigators in the proposed program offer an unusual opportunity to define the molecular mechanisms responsible for neurodegeneration. Elucidation of the mechanisms by which brain cells cease to function and die in prion disease after a long delay may offer new approaches to elucidating the etiologies of more prevalent neurodegenerative disorders afflicting other people, including AD.
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0.936 |
1992 — 2001 |
Prusiner, Stanley B |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Experimental Approaches to Neurodegenerative Diseases @ University of California San Francisco |
0.936 |
1993 — 2009 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Molecular Pathogenesis of Age-Dependent Cns Degeneration @ University of California San Francisco
CNS degenerative diseases are among the most dreaded afflictions from which older people frequently suffer. The most common of these neurodegenerative disorders is Alzheimer's disease. Studies on the etiologies of both the familial and sporadic forms of Alzheimer's disease have been frustratingly slow in part due to our lack of both knowledge about its molecular pathogenesis and suitable animal models. In contrast, the availability of animal models for neurodegenerative diseases caused by prions has led to relatively rapid advances. Indeed, prions diseases have become the most well understood CNS degenerative disorders of delayed onset and they are the focus of this proposal. In humans, prion diseases generally occur in older adults but the mechanism governing their time of onset is unknown. We plan to identify neurotransmitter/receptor systems that malfunction in both the genetic and infectious forms of prion diseases. Alterations in brain lipids, glycolipids and activated glycolipids and activated glycolipids will be measured since their regulation is intimately linked to the synthesis of the prion protein (PrP) which is a sialoglycoprotein that carries a glycoinositol phospholipid (GPI). Measurements of Ca++ flux and compartmentalization will be studied in scrapie-infected cultured cells where abnormalities in Ca++ metabolism have already been identified. Alterations in Ca++ metabolism have been demonstrated in recent years to feature prominently in CNS dysfunction and neuronal death. While considerable progress has been made in deciphering the cellular processes which govern prion incubation times, relatively little is known about the molecular mechanisms responsible for distinct prion isolates or "strain". Transgenic mice crossed with mice in which the murine PrP gene has been ablated will be used to define those factors which govern the phenotype of the "infectious" prions. Physical studies designed to search for a hypothetical cellular RNA which has been proposed to modify the properties of PrPSc will be performed as will studies on the conformation of PrPSc in two isolates exhibiting different incubation times. These proposed studies address the molecular mechanism responsible for the pathogenesis of both the infectious and genetic forms of prion diseases and by inference the most common form of CJD which is sporadic. The diverse skills, talents and backgrounds of the investigators in the proposed program offer an unusual opportunity to attack the molecular mechanisms underlying the neuropathogenesis of prion diseases. Elucidation of the mechanisms by which brain cells cease to function and die in prion diseases after a long delay may offer approaches to under standing how neurons develop, mature, continue to transmit signals for decades and then become senescent. Moreover, understanding prion diseases may give valuable new insights with respect to the etiologies of more common CNS degenerative disorders afflicting older people, including Alzheimer's disease.
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0.936 |
1994 — 2002 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core-- Animal @ University of California San Francisco
Bioassays of prions and replication of prions to high titers must still be performed in animals. Generally, laboratory rodents can be used. Transgenic mice expressing foreign and mutant PrP genes have revolutionized prion research. A highly efficient Animal Core is required for the studies describes in this application. The specialized personnel, facilities, techniques and procedures required for prion research have been developed over the past 15 years at UCSF. The Animal Core is directed by Dr. Marilyn Torchia, a veterinarian with many years of experience in the care of laboratory animals. Transgenic mice will be analyzed for scrapie incubation times after inoculation with prions, while others which are uninoclated will be observed for the onset of spontaneous disease. Besides measuring incubation times, end point titration of prions will be measured for a small number of selected samples. Prions will be replicated to high titers in Syrian hamsters. After these hamster develop clinical signs of neurologic dysfunction, they will be sacrificed and their brains removed. From frozen hamster brains, prions will be purified as described in the Scientific Core. To insure accurate record keeping and to facilitate all phases of the Animal Core operations, mice are tattooed with a number which corresponds to a bar-code on the cage card. Bar-code reader are used to enter all data on the status of each animal. Data is then downloaded to a computer and analyzed using a database program. We anticipate that our computerized animal care management system will become even more important as transgenic mice are mated with mice carrying ablated PrP alleles. Because of the prolonged incubation times in prion diseases, many experiments must be done in parallel. Economic and physical laminations on the size of the Animal Core necessitate careful planning and frequent reevaluation of priorities. In order to maximize the number of animals that are evaluated, cost containment and reduction are constant objectives. The specialized capabilities and services of the Animal Core described here are crucial for all phases of prion research.
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0.936 |
1994 — 2009 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Animal Core @ University of California San Francisco
The function of the Animal Core is to produce and maintain all of the animals required for the scientific projects for this and several other Program Project grants. The specific services provided by the Animal Core personnel include: (1) production of transgenic and knockout mice; (2) production of large volumes of prion-infected hamster and mouse brains for purification; (3) performing experimental inoculations, neurologic scoring of animals, data collection, and tissue collection; (4) providing transportation of animals and tissues between the laboratory and the animal facility; (5) production of antibodies in mice and rabbits for experimental use; (6) providing all animal care and veterinary care; (7) providing cryopreservation of the various mouse lines. The Animal Core operates in two purpose-built facilities in the Hunters Point area of San Francisco, located approximately 3 miles from the main UCSF campus. The activities of the Animal Core are directed by Dr. Prusiner and Dr. Pierre Lessard, a laboratory animal veterinarian. Building 830B is a new nine-room transgenic mouse breeding facility that houses our large breeding colony. Building 830 houses all the experimental animals in 21 rooms, under BSL-2 and BSL-3 biocontainment. It houses, on average, 20,000 mice, 400 hamsters, and 3 rabbits. The facility is inspected bi-annually by the USDA and UCSF/IACUC.
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0.936 |
1994 — 1999 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Defining the Molecular Basis of Prion Diversity Through Transgenetics @ University of California San Francisco
Many experimental approaches have failed to find evidence for a scrapie- specific nucleic acid, yet the identification of distinct scrapie "strains" challenges this conclusion. Studies of prion strains have been facilitated by transgenic (Tg) mice since overexpression of PrP leads to a pronounced shortening of the incubation time. Use of mutated or chimeric PrP transgenes provides a source of PrP genetic variation not possible by other means. Syrian hamsters (SHa) have proven to offer significant advantages over mice for purification of infectious prions since SHa contain higher prion titers and 10-50-fold more PrPSc than mice making them more suitable for purification of PrPSc for structural studies. Tg mice expressing a chimeric SHa/Mo PrP gene designated MH2M are highly susceptible to prions derived from both mice and SHa. We propose to transfer distinct mouse prion strains into SHa using Tg(MH2MPrP) mice as an intermediate host. We have already made progress toward this goal and have obtained SHa(RML), SHa(Me7) and SHa(22A) prion inocula. We shall continue these studies by passaging of the 87V and 301V mouse strains from Tg(MH2MPrP) mice into SHa giving us five Mo- derived SHa prion isolates in addition to the SHa prion strains: Sc237, 139H, MT-C5, DY and Me7H. The strain Me7H is clearly distinct from Me7 derived from Tg(MH2MPrP) mice. The potential usefulness of these SHa strains will depend on several factors, including stability upon repeated passage, and our ability to assign discrete characteristics. Once two to more distinct SHa prion strains have been identified and large quantities of stock inocula have been prepared, we shall purify the prions and analyze the PrPSc using FTIR and CD spectroscopy, surface plasmon resonance, deuterium exchange as probed by mass spectrometry, chaotrope mediated denaturation in concert with bioassays, protease digestion and electron microscopy. Additional studies will also employ mass spectrometry in an effort to identify any additional components which might be responsible for scrapie prion diversity. To exploit further the advantages of the SHa, we plan to produce Tg SHa overexpressing SHaPrP and to derive SHa embryonic stem (ES) cells in which the endogenous SHaPrP gene has been disrupted. These ES cells will be used to produce chimeric SHa which will be crossed to produce PrP-o/o SHa. Such null SHa may be ideal hosts for expressing high levels of mutant and chimeric PrP transgenes, as well as genes derived from other species. To facilitate purification of PrPSc from Tg SHa and mice, affinity "tagged PrP" will be expressed once we show that it can be converted into PrPSc and supports prion replication. Deciphering the molecular basis of prion strains promises to open many avenues of research on prions as well as information transfer between proteins.
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0.936 |
1994 — 1996 |
Prusiner, Stanley B |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Leadership and Excellence in Alzheimers Disease @ University of California San Francisco
Alzheimer's disease (AD) is a rapidly growing problem of epidemic proportions. The cause of AD is unknown. The lack of a "perfect" animal model for AD has slowed progress in deciphering its etiology. While experimental scrapie in animals differs in some respects from AD, many aspects of the CNS degeneration caused by scrapie prions resembles that found in AD. We propose to exploit some recent, unprecedented discoveries in prion diseases of animals and humans. Molecular genetic studies in mice and humans have shown that susceptibility to prion diseases - experimental scrapie in mice and Gerstmann-Straussler syndrome (GSS) in humans - is genetically linked to mutations in the open reading frame (ORF) of the prion protein (PrP) gene. We plan to sequence the ORF's of PrP genes from patients with GSS and familial Creutzfeldt-Jakob disease (FCJD) defining the extent of human PrP gene variation. Additional linkage studies with new PrP gene mutations in families with GSS of FCJD will be undertaken. Sequencing of entire PrP genes from normal humans, patients of GSS, inbred mice with long and short incubation times, sheep breeds with different susceptibilities to scrapie and several species of hamsters with distinct incubation times is planned. A search for prion diseases involving proteins other than PrP is proposed. Grey tremor mice will be examined first since it appears to be both genetic and infectious like GSS. We plan to create physical maps for mice and humans extending about 5 Mb on each side of the PrP gene. YAC clones will be recovered and used to construct contig maps. Probes derived from these YAC clones will be employed to identify a linked scrapie incubation time gene (Prn-i) in mice and the GSS gene in humans if these prove to be separable from the PrP gene. We plan to identify PrP related genes in mammals and in lower organisms especially those amenable to genetic manipulation. As more is learned about prion diseases it may be possible to determine whether natural scrapie is a genetic or infectious disease - both mechanisms have been proposed. We plan a set of genetic linkage studies in sheep with scrapie. Random mutagenesis of PrP genes from rodents will be performed and expressed clones assessed for production of PrPSc and infectivity. Such studies may ultimately elucidate the mechanisms underlying diseases which are both infectious and genetic. Indeed, unprecedented discoveries in studies on prion diseases signal new directions in CNS degenerative disease research;the implications for Alzheimer's disease and some other neurodegenerative disorders of older people may be profound.
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0.936 |
1994 — 2020 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Degenerative and Dementing Diseases of Aging @ University of California San Francisco
Dementing diseases are among the most dreaded afflictions from which older people frequently suffer. Alzheimer's disease (AD) is the most common dementia. AD is a neurodegenerative disorder, the etiology of which is unknown expect for some familial cases. Studies on both the familial and sporadic forms of AD have been slow, in part, due to our lack of suitable animal models. In contrast, the availability of animal models for neurodegenerative diseases caused by prions has led to relatively rapid advances. Indeed, prion diseases have become the most will understood CNS degenerative disorders of delayed onset and they are the focus of this proposal. In humans, prion diseases generally occur in older adults but the mechanisms governing their time onset is unknown. Recent studies argue that prion diseases may be disorders of protein conformation. Investigations directed toward elucidating the mechanism of conversion of the cellular prion protein (PrPc) into the scrapie isoform (PrPsc) are proposed. Experimental and theoretical studies on the secondary, tertiary and quartenary structures of PrPc and PrPsc as well as synthetic PrP peptides are planned. To facilitate these investigations, recombinant antibodies to PrPc and PrPsc will be produced which can discriminate between the different conformations of PrP. Studies on the cell-free synthesis of PrP are designed to develop systems for the in vitro generation of PRPSc in conjunction with studies on the renaturation of PrPsc. Besides using conformational dependent PrP antibodies to study PrPsc renaturation and cell-free synthesis, they will be used to investigate the patterns of PrPSc accumulation in sporadic and familial Creutzfeldt-Jakob disease (CJD). Each distinct isolate or "strain" of scrapie prions has unique pattern of PrPSc accumulation suggesting that the molecular basis of prion diversity resides in the cell-specific synthesis of PrPSc accumulation suggesting that the molecular basis of prion diversity resides in the cell-specific synthesis of PrPSc. The largest focus of familial CJD cause by a PrP codon 200 mutation is found in Libyan Jews living in Israel. Patients as well as at risk and unaffected family members of Libyan Jews will be studied. The investigations proposed here address the molecular mechanism responsible for the pathogenesis of the infectious, genetic and sporadic forms of prion diseases. The diverse skills, talents and background of the investigators in the proposed program offer an unusual opportunity to define the molecular mechanisms responsible for neurodegeneration in prion diseases. Elucidation of the mechanisms by which brain cells cease to function and die in prion diseases after a long delay may offer new approaches to elucidating the etiologies of more prevalent neurodegenerative disorders afflicting older people, including AD.
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0.936 |
1995 — 1999 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Molecular Transgenetics of Human Prion Disease @ University of California San Francisco
Recent successful investigations into CNS degeneration caused by prions demand that some striking observations be exploited. To take advantage of these discoveries, we propose to develop and extend several new and innovative approaches to transmissible neurodegenerative diseases utilizing molecular genetics, transgenic animals and cultured cells. Molecular genetic studies in mice and Gerstmann-Straussler syndrome (GSS) in humans - is genetically linked to polymorphisms in the open reading frame of the prion protein (PrP) gene. These polymorphisms result in nonconservative amino acid substitution.s Linkage of a PrP missense variant to the development of GSS established that GSS is a genetic disease which is also infectious and raises the possibility that the leucine variant at PrP codon 102 causes GSS. Recently, we have found that transgenic (Tg) mice inoculated with Ha scrapie prions exhibit scrapie infectivity, incubation times and PrP amyloid plaques characteristic of hamsters. These studies are the first demonstration of the synthesis of infectious scrapie prions programmed by a recombinant DNA molecule; furthermore, they have created a shift in prion research from an area restricted to descriptive and correlative observations to one now amenable to experimental manipulation. We propose to determine whether a GSS mutant PrP transgene will spontaneously produce scrapie in mice. The GSS mutant PrP will also be expressed in human cells to determine whether prions are produced spontaneously. We shall use Tg mice with chimeric Ha/Mo transgenes to identify specific domains of HaPrP which are required for Ha prion infectivity synthesis. Additional studies will focus on cultured cells for the synthesis of PrPSc from expression vectors and use of these cells to define the steps of PrPSc synthesis. Considerable effort will be devoted to attempts to develop a new bioassay for prions in cultured cells. We shall also try to develop a system for cell-free synthesis of PrPSc; such a system should lead to the purification of macromolecules involved in the conversion of PrPC or a precursor into PrPSc. The unprecedented discoveries in studies on prion diseases signal new directions in CNS degenerative disease research and may have profound implications for understanding a variety of neurodegenerative processes, most notably some disorders of unknown etiology such as Alzheimer's disease.
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0.936 |
1995 — 2008 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core--Animal @ University of California San Francisco
The function of the Animal Core is to produce and maintain all of the animals required for the scientific project for this and several other program project grants. The specific services of the Animal Core personnel include 1) Production of transgenic and knockout mice, 2) Production of large volumes of scrapie-infected hamsters and mouse brains for purification, 3) Performing experimental inoculations, neurologic scoring of animals, data collection, and tissue collection, 4) Providing transportation of animals and tissues between the laboratory and the animal facility, 5) Production of antibodies in mice and rabbits for experimental use, 6) Providing all animal care and veterinary care, 7) Providing cryopreservation of the various mouse lines. The Animal Core operates in two purpose-built facilities in the Hunters Point area of San Francisco, approximately 3 miles from the main UCSF campus. The activities of the Animal Core are directed by Dr. Prusiner and Dr. Pierre Lessard, a laboratory animal veterinarian. Building 830B is a new nine room transgenic mouse breeding facility that houses our large breeding colony. Building 830 houses all the experimental animals in 21 rooms, under BSL-2 and 3 biocontainment. It houses on average 10,000 mice, 400 hamsters, and 8 rabbits. The facility is inspected annually by USDA, and bi-annually by UCSF/IACUC.
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0.936 |
1995 — 2002 |
Prusiner, Stanley B |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Core--Animal Core @ University of California San Francisco
Our research program on transmissible and genetic neurodegenerative diseases rests upon the Animal Core. The need for propagation of prions to high titers and for bioassay of prion infectivity requires extremely large numbers of mice and hamsters. Without proper animal facilities, personnel and support, the studies described to date could not have been performed, and the studies outlined in this proposal cannot be carried out. Over the past fifteen years we have accumulated a large amount of experience with hamsters and mice infected with scrapie prions. This experience has helped us to delineate the minimum requirements for operating a successful facility. We have a well-trained staff of animal caretakers directed by skilled supervisors and a veterinarian. Many steps are involved in the care of animals inoculated with prions. The inoculations must be done with the greatest of skill. The animals must be given exquisite animal care and then they must be checked with great frequency and the diagnosis of scrapie made with extreme reliability. Careful records must be kept and selected animals must be examined for neuropathologic evidence of scrapie. With the recent development of transgenic mice expressing foreign PrP genes, the number of animals and the complexity of the record keeping in the animal core will increase since each new transgenic line represents a new strain of mouse. We are very fortunate to have a very skilled veterinarian, Dr. Marilyn Torchia, directing the animal facility operation and to have the University of California facilities available to us at Hunters Point in San Francisco. The details of the animal care program are described in Core A, as well as the animal usage for each scientific project.
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0.936 |
1995 — 2002 |
Prusiner, Stanley B |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Core--Scientific Core @ University of California San Francisco
The Scientific Core is a modest unit set up to facilitate the scientific projects within the program. The services and reagents provided by the Scientific Core are purified prions, antibodies, synthetic peptides, synthetic oligonucleotides and neuropathologic evaluations. Most of the projects require purified prions, bioassays, antibodies to PrP peptides, synthetic oligonucleotides and neuropathologic evaluations of selected animals. Three of the projects also use synthetic peptides. To provide these services and reagents, Dr. Stephen DeArmond, a skilled neuropathologist and a talented technician, are needed. The technician will ensure the smooth operation of the Scientific Core and the provision of all reagents and services.
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0.936 |
1995 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Molecular Pathogenesis of Age Dependent Cns Degeneration @ University of California San Francisco |
0.936 |
1995 — 1999 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Transmissible and Genetic Neurodengenerative Diseases @ University of California San Francisco
Prion diseases are neurodegenerative disorders of humans and animals. These diseases can present as genetic, infectious or sporadic illnesses and are often transmissible to experimental animals. Convergence of many lines of experimental evidence asserts that the scrapie isoform of the prion protein (PrP/Sc) is a necessary component of the infectious scrapie prion yet both PrP/Sc and the normal isoform, PrP/C, are encoded by the same chromosomal gene. All attempts to identify a candidate scrapie- specific nucleic acid in fractions enriched for scrapie infectivity have failed. Seven senior investigators with outstanding records of accomplishment in a variety of scientific disciplines and with demonstrated commitments to scrapie research propose to extend their collaborative studies currently supported by an NINDS-Program Project Grant. The exciting progress in scrapie research over the past five years coupled with the complementary talents and skills of this group of scientists represents a unique opportunity to make further advances by taking advantage of some unprecedented discoveries in prion diseases. Molecular genetic studies in sheep, mice and humans have shown that susceptibility to prion diseases--natural scrapie of sheep, experimental scrapie in mice as well as Gerstmann-Straussler (GSS) and Creutzfeldt- Jakob diseases (CJD) in humans--is genetically linked to amino acid substitutions in the open reading frame of the prion protein (PrP) gene. Studies with congenic and Tg mice expressing different numbers of the a and b alleles of mouse (Mo) PrP have shown that long incubation times are not dominant traits but rather result from the effects of PrP gene dosage. These findings overturned one of the major tenets which were established in scrapie research over the past 25 years. Highly sensitive techniques for detecting low concentrations of nucleic acids have been developed but no scrapie specific candidate polynucleotide has been found. These results are of considerable importance, as the number of hypotheses available to explain strains of prions continues to shrink. Construction of chimeric transgenes has given us a new system for the study of the human (Hu) prion diseases; Tg(MHu2M) mice expressing the chimeric Hu/Mo PrP are susceptible to Hu prions. The infrequent transmission of prion disease to Tg(HuPrP) mice in contrast to Tg(MHu2M) mice argues that a species-specific factor which we have provisionally designated as protein X governs the conversion of PrP/C into PrP/Sc. Whether protein X is molecular chaperone remains to be determined. Chimeric and tagged PrP/Sc molecules will be employed in the studies designed to unravel the enigma of prion strains. The molecular basis of prion "strains" presents a fascinating conundrum; studies of strains may be facilitated by using chimeric PrP molecules, especially if different prion strains prove to be distinct conformers of PrP/Sc. Our studies may ultimately elucidate the mechanisms responsible for a variety of CNS degenerative disorders, in particular those of unknown etiology afflicting older people such as Alzheimer's disease.
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0.936 |
1997 — 2011 |
Prusiner, Stanley B |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Structural Characterization of Prion Proteins @ University of California San Francisco |
0.936 |
1998 — 2002 |
Prusiner, Stanley B |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Nmr Structures of Recombinant Prps @ University of California San Francisco
The major goals for this project are the structural elucidation of soluble PrP fragments using NMR spectroscopy. It is clear from both biological and preliminary studies that the region of the PrP/c molecule that lie close to the N-terminus are potentially of great significance in the structure and function of the PrP, and in the alpha-->beta conversion that is thought to be the source of prion disease and infectivity. The first specific aim will concentrate on the determination of the structure of the H1 and octarepeat regions of the SHaPrP/c protein, in the context of sizeable fragment containing regions that are known to be folded in solution. Similarity between the NMR spectra of the full-length construct 29-231 and a shorter fragment 90-231 indicate that the longer fragment may have a domain structure, but a significant increase in stability is also indicative that the H1 and octarepeat regions have structural significance. Binding of metal ions of possible physiological significance will be investigated. Preliminary 3D triple resonance NMR spectra of the 29-231 fragment are excellent, and give great promise that resonance assignments and solution structure determination will be possible in this challenging system. It is of considerable importance and interest to discover the structural basis for the alpha-->beta conversion. To this end, the second and third specific aims will address the structure of wt and mutant chimeric MH2M PrP fragments, point mutants A117V and P102L and a triple A113V, A115V, A118V mutant. The mutant residues are in the center of the region that is vital to the conformational plasticity of the N- terminal section of PrP, and the point mutants have been implicated in familial prion disease. Structural studies on these proteins should allow us to probe the nature of the alpha-->beta conversion. Certainly the change of three helix-promoting Ala residues for three beta-promoting Val residues would be expected to favor the veta form of the protein. Circular dichroism studies of the 90-231 recombinant triple mutant show substantial beta-sheet structure and much less alpha-helix. In an exciting new development, a 106-residue PrP fragments has been prepared where several loops of the intact protein have been deleted. This fragment is the first soluble PrP/sc-like molecule and represents our best opportunity to learn about the structure of the disease-causing isoform. The fourth specific aim therefore addresses structure determination by NMR of this fragment. The specific aims are closely related to each other and to the goals of the overall program project. They address specifically the structural basis of the formation of prions by structure elucidation of folded fragments in solution.
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0.936 |
1998 — 2002 |
Prusiner, Stanley B |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Transgenic Studies of Mutant Prp Genes @ University of California San Francisco
Prion diseases are disorders of protein conformation. The pathogenic form of the prion protein, PrP/sc, is distinguished from its cellular counterpart, PrP/C, by a marked increase in the degree of beta-sheet content and a decrease in the proportion of alpha-helix. Although progress has been made in efforts to determine the three-dimensional structure of PrP/c, considerable work remains. Even more challenging is determining the structure of PrP/sc, which is extremely insoluble in the native state. The insolubility of PrP/sc has hindered attempts to elucidate the complete molecular structure of the infectious prion. To gain insight into the structural transitions which feature in PrP/sc formation, we plan to create mutations in PrP which will facilitate structural studies of PrP/sc. Our goal is to create novel mutations which lower the activation energy barrier for spontaneous formation of infectious prions de novo, especially using recombinant PrP in an in vitro reaction. By using computational methods, we have shown that it is possible to design highly pathogenic mutations in PrP which cause disease with unprecedented rapidity. Preliminary studies indicate that transmissible prions may be formed de novo, and spectroscopic studies have shown that the mutant protein adopts a beta-sheet conformation under conditions where the wild- type protein is predominantly alpha-helical. Of major importance, the beta-sheet conformer formed in vitro is soluble and preliminary NMR studies have been initiated. In a complementary approach, we have identified a mutated PrP/c molecule of 106 residues that can be converted into a protein that closely resembles PrP/sc when expressed in scrapie- infected mammalian cells. This truncated PrP/sc-like molecule is soluble in the presence of low concentrations of ionic detergent, facilitating purification of the protein for structure determination. In this proposal, we describe experimental studies which exploit these findings and extend out knowledge or PrP/sc formation. Such investigations in concert with those outlined in Projects 2, 3 and 4 should allow us to determine the complete molecular structure for a prion.
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0.936 |
1999 |
Prusiner, Stanley B |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Structural Characterization of Prion Proteins: Creutzfeld Jakob Dis, Scrapie @ University of California San Francisco
Introduction Prion diseases are unique fatal neurodgenerative diseases caused by novel pathogens termed prions. The major or only component of prions is an abnormal form of a normal cellular protein termed the prion protein (PrP). The normal form (PrPC) is converted to the pathogenic form (PrPSc) by an as yet unidentified posttranslational process. Mass spectrometry is essential for the detailed analysis of small-scale samples of both forms of the prion protein isolated from the brains of hamsters. methods: Several novel methods have been refined for the analysis of PrPSc using LSIMS, ESIMS, MALDI and MS/MS for the characterization of peptides and oligosaccharides from enzyme digests. These analyses are complicated by the hydrophobic, insoluble and strongly aggregating propoerties, particularly of PrPSc. PrPC analyses have been delayed by problems of purifying the protein from brain. Preliminary results by MALDI show that the two forms are similar, but microheterogeneity has prevented any definitive conclusions being drawn. Comparisons are also required between PrPSc from different prion strains. Strain behavior could be due to differences in the N-linked oligosaccharides; perhaps cell specific lectins cause targetting of certain PrP species to a limited range of cell types, resulting in regional distribution of PrPSc. We plan to use LC/MS for much of this characterization. Discussion: Results obtained so far suggest that the modification of PrPC involved in the formation of PrPSc may be conformational rather than chemical. It is essential to confirm that PrpC and PrPSc either are or are not identical in order to develop hypotheses for the molecular mechanisms of prion diseases.
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0.936 |
1999 — 2002 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Purification and Structural Studies On Prpsc106 @ University of California San Francisco
Progress in studies on the structural transition of PrPc into PrPSc requires soluble PrPSc. Toward this goal, we propose to exploit an exciting set of findings where approximately 50% of the PrP molecule was deleted and yet the mutant protein could be converted into PrPSc. This mutant PrP contains 106 residues and is denoted PrP106 while the pathogenic isoform is designated PrPS106. In scrapie infected mouse neuroblastoma (ScN2a) cells, PrPSc106 is protease-resistant and can be solubilized in 1% Sarkosyl under conditions that are known to preserve prion infectivity. Furthermore, Tg (PrP106)Prnp0/0 mice developed signs of CNS dysfunction at approximately 65 days after inoculation with RML106 prions. We propose to characterize prions containing PrPSc106, to develop suitable large-scale purification procedures, and to perform structural studies of PrPSc106. Standard curves for bioassay of RML106 prions will be constructed. Optimal conditions for solubilizing PrPSc106 from transfected ScN2a cells, brains of Tg(PrP106) Prnp0/0 mice and cultured cells derived from embryos PrPSc106, we shall identify suitable hydrophobic residues on the surface of PrP106 that can be substituted with hydrophilic residues. A combinatorial strategy will be employed to mutate several sites simultaneously, and a screening protocol using an automated robotic workstation employed. N- terminally modified PrP106 will be tested for increasing the rate of PrPSc106 formation and elevating the levels of this pathogenic isoform. The charged N-terminal residues will be used as an epitope for generating anion- dependent antibodies that could be used in a high yield, affinity purification scheme. Multiple strains of PrP106 prions will be produced by passage in Tg(PrP106)Prnp0/0 mice if the physical properties of PrPSc106 from any strain are more amenable to physical characterization than those of PrPSc106 from RML106.
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0.936 |
2000 — 2005 |
Prusiner, Stanley B |
M01Activity Code Description: An award made to an institution solely for the support of a General Clinical Research Center where scientists conduct studies on a wide range of human diseases using the full spectrum of the biomedical sciences. Costs underwritten by these grants include those for renovation, for operational expenses such as staff salaries, equipment, and supplies, and for hospitalization. A General Clinical Research Center is a discrete unit of research beds separated from the general care wards. N01Activity Code Description: Undocumented code - click on the grant title for more information. |
Development of Assay For Creutzfeldt-Jakob Disease @ University of California San Francisco |
0.936 |
2000 — 2002 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Biochemical and Transgenic Studies of Prion Strains @ University of California San Francisco
Prion strains can be distinguished by differences in incubation times, distribution of spongiform pathology, and the pattern of PrPSc deposition. For many years the existence of prion strains. For many years the existence of prion strains was used as an argument for the existence of an essential, as yet unidentified, nucleic acid in the prion particle. During the past five years, considerable evidence has accumulated arguing that the properties of prion strains are enciphered in the conformation of PrPSc. Using a conformation-dependent immunoassay (CDI), we detected differences in the tertiary structures of PrPSc for either different strains. Moreover, the levels of protease-sensitive PrPSc were found to correlate with the length of the strain specified incubation time. We plan to purify both protease-sensitive and-resistant PrPSc from the brains of Syrian hamsters infected with different prion strains and to compare the physical and biological properties of these PrP isoforms. Whether protease- sensitive PrPSc carries prion infectivity of whether it can be converted into protease resistant PrPSc remains to be established. Since studies of protease-sensitive PrPSc support the proposal that PrPSc clearance may determine the length of the incubation time, we plan to determine the rates of formation and clearance for both protease-sensitive and -resistant PrPSc. To do this, bigenic mice expressing tetracycline-regulated PrP transgenes that are inoculated with different strains will be studied. To assess the relative roles of conformation and glycosylation in the enciphering of strain specified properties, we propose to construct transgenic mice expressing glycosylation in the enciphering of strain specified properties, we propose to construct transgenic mice expressing glycosylation deficient PrPs and determine if they are competent for replication of different prion strains. With Gln substitutions at Asn-linked initiated the formation of unglycosylated PrPSc. In this proposal, we introduce the concept that prion strain variation might arise from two sources: (1) qualitative differences in conformation of PrPSc or (2) quantitative variations in the ratio of discrete conformers. In some cases, strains with similar biological characteristics may be distinguished primarily by variations in the ratio of conformers. In other cases, particularly where mor extreme phenotypic differences are observed, distinct conformers of PrPSc may be identified.
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0.936 |
2001 |
Prusiner, Stanley B |
C06Activity Code Description: To provide matching Federal funds, up to 75%, for construction or major remodeling, to create new research facilities. In addition to basic research laboratories this may include, under certain circumstances, animal facilities and/or limited clinical facilities where they are an integral part of an overall research effort. |
Biosafety Laboratory (Bsl-3) Studies Human/Bovine Prions @ University of California San Francisco |
0.936 |
2003 — 2013 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Novel Therapeutics For Prion Diseases @ University of California San Francisco
DESCRIPTION (provided by applicant): Prion diseases are neurodegenerative diseases of humans and animals, which are invariably fatal and lead to death within a year after the onset of clinical symptoms. As with most other neurodegenerative diseases, no effective therapy is known. Treatment of patients with sporadic (s) Creutzfeldt-Jakob disease (CJD), the most common human prion disease, employing quinacrine will be studied. CJD patients will initially receive a racemic mixture of quinacrine. Quinacrine has been shown to inhibit prion formation in cultured ScN2a cells at submicromolar concentrations. Additionally, experimental prion disease in mice will be treated with quinacrine. In studies with ScN2a cells, the (S)-quinacrine isomer was 2 to 3 times more potent with respect to inhibiting prion formation than (R)-quinacrine; these isomers will be compared to the racemic mixture in mice. Concurrently, murine models will be used to evaluate treatment of prion disease with new drugs produced through empirical and rational drug design. The empirical drug program will utilize a combinatorial chemistry approach with quinacrine as the lead compound. Quinacrine will be modified and tested in the ScN2a cell culture system. We plan to test about 2000 new quinacrine analogs per year. Only compounds that are 10 times more potent than quinacrine with respect to antiprion activity will be evaluated in mice. Initially, these new antiprion compounds will be screened for toxicity. Compounds that are determined to be sufficiently nontoxic will then be tested for their ability to block prion synthesis in transgenic (Tg) mice. Besides empirical drug discovery, we plan to expand a rational drug design program along two lines of investigation. Attempts will be made to increase the potency of quinacrine by further modifying the aliphatic side chain. Recent studies have shown that bis-quinacrine analogs are more potent than quinacrine by a factor of ten. We also plan to dissect the mode of quinacrine action through studies of PrP trafficking in cultured cells. A second line of rational drug design involves modifying compound 60, which was found by mimicking dominant negative inhibition of prion synthesis. In order to understand dominant negative inhibition of prion synthesis, the structures of dominant negative PrPs will be determined using NMR spectroscopy. The information obtained from these dominant negatives should facilitate improvements in the design of existing drugs or lead to the production of new drugs. PRINCIPAL INVESTIGATOR The PI of this PPG, Dr. Stanley Prusiner, is a professor of Biochemistry and the director of Neurodegenerative diseases at UCSF. He has extensive experience in the area of virology and neurology and in directing research projects of this magnitude. He is a leading authority in prion research for the last several years. He is the recipient of numerous national and international awards, including the nobel prize in 1998. He has published more than 200 articles in scientific journals of interanational repute. He has trained and supervised several researchers/clinicians and thus is well qualified to lead this group and the project. REVIEW OF INDIVIDUAL COMPONENTS PROJECT 1: Clinical Study of Quinacrine for Treatment of Human Prion Diseases; Dr. Richard Miller (PL) DESCRIPTION (provided by applicant): Creutzfeldt-Jakob disease (CJD)is a rapidly progressive, invariably fatal and untreatable neurodegenerative disease with a mean duration of about eight months. Beyond the debilitating cognitive and motor deficits that accompany CJD, the difficulty in treating behavioral and mood disturbances and the rapidity of its course compound its tragedy. Moreover, an epidemic of new variant CJD (nvCJD) in England has raised serious concerns regarding the safety of the world's beef supply; the possibility that prions might be passed through the blood has led to the banning of blood or tissue donations from individuals who have resided in England. The discovery of an effective therapy for prion diseases would have enormous human and economic implications. Recent results from experiments in Dr. Prusiner's laboratory show that, at physiological concentrations, the anti-malarial drug quinacrine permanently clears abnormal prion proteins from cell culture. The demonstrated efficacy of quinacrine in cell culture, its relative safety and well known side-effects in the clinical setting, and the universal fatality of CJD justify quinacrine as an immediate candidate for the treatment of CJD. We propose a treatment study for patients with sporadic CJD (sCJD) with racemic quinacrine. Over three years, 90 patients will be admitted to the University of California at San Francisco (UCSF) NIH-funded clinical research center where a diagnosis of sCJD will be determined and where patients will enter into a randomized, double-blinded, treatment study with quinacrine. Patients will be divided into two quinacrine arms, a high-dose titration (450 mg daily) and a low-dose titration (75 mg daily). They will be treated for one year and then be followed through to the end of the five-year study period. The dose of quinacrine may be increased or decreased in each patient depending on clinical deterioration or toxicity, respectively. Survival will be the primary outcome measure of this clinical study. Also, additional outcome measures will be used that assess activities of daily living, cognition, MRI and EEG. We hypothesize that patients in the high-dose quinacrine arm will have increased survival and a slower rate of neurological progression compared with patients in the low dose arm. By year four of this program Project grant (PPG), we hope to begin a clinical study with a new compound, developed in other Projects in this PPG that shows even greater efficacy than racemic quinacrine.
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0.936 |
2004 — 2005 |
Prusiner, Stanley B |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
The Search For Protein X Using Crosslinking and Other Approaches @ University of California San Francisco |
0.936 |
2004 — 2006 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Molecular Basis of Species Barrier of Prion Transmission @ University of California San Francisco
We propose to decipher the molecular basis of the species barrier by using transgenic (Tg) mice expressing chimeric human (Hu)/mouse (Mo) PrP genes. Earlier studies showed that mice expressing a chimeric PrP transgene designated MHu2M were susceptible to Hu prions. After systematically reverting some of the nine Hu residues to Mo at the N- and C-terminal ends of the "Hu2" insert, we identified Tg(MHu2M,S96N) mice with prolonged incubation times and Tg(MHu2M,M165V, E167Q) mice with abbreviated incubation times. The C-terminal residues 165 and 167 appear to be important in the binding of chimeric PrP to an auxiliary protein, provisionally designated protein X, that features in prion replication, while the N-terminal residues appear to bind to PrPSc in the inoculum. Our findings define a limited but feasible study of residues 96, 165 and 167 as well as the remaining six Hu residues in the Hu2 insert. Within the constraints posed by the need to construct Tg mice, we propose to introduce a substantial number of changes at the positions of the nine Hu residues in the Hu2 insert. Individual substitutions as well as combinations in the form of doublets and triplets will be made. We also plan to develop a cell culture system for the bioassay of chimeric PrP genes in ScN2a and ScGT1 cells by disrupting both copies of the MoPrP gene in these cells as described in Project 13. Such an assay would lift the constraints posed by the current need for Tg mice to test each chimeric PrP gene construct. From our studies of Tg mice, we should be able to determine the effects each residue in the Hu2 insert on incubation times for Hu prions and apply this knowledge to the development of rapid Tg mouse models for both Hu prion diseases and Chronic Wasting Disease (CWD) in cervids.
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0.936 |
2004 — 2005 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Administrative Core @ University of California San Francisco |
0.936 |
2005 — 2009 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
De Novo Generation of Mammalial Prion Strains @ University of California San Francisco
A wealth of data argues that mammalian prions are composed of infectious proteins designated PrPSc. Encouraged by investigations demonstrating the production of synthetic prions was possible by mutating the prion protein (PrP) gene, we chose to study a subset of recombinant (rec) PrPs folded into beta-rich structures. PrP amyloid is formed from N-terminally truncated PrPSc referred to as PrP 27-30, which comprises residues 89 to 230 in mouse (Mo) PrP and retains prion infectivity. Using only recMoPrP(89-230) produced in E. coli to form amyloid fibrils, we inoculated the fibrils intracerebrally into transgenic (Tg) mice expressing MoPrP(delta-23-88) (Supattapone et al. 2001). After more than 300 days, none of the mice had developed symptoms of CMS disease; therefore, we reported that the N-terminally truncated MoPrP(89-230) folded into amyloid fibrils were not infectious. Subsequently, we found that all of these mice developed neurologic dysfunction between 380 and 660 days after inoculation. Western blotting of brain extracts showed protease-resistant PrP and serial transmission of the prions to wild-type (wt) FVB and Tg(MoPrP)4053 mice gave incubation times of 150 and 90 days, respectively. Neuropathological examination suggests that a novel prion strain was created, one that is distinct from the widely used RML strain. Our discovery supports the propositions that prions are infectious proteins and that sporadic prion disease requires only the spontaneous conversion of PrPC into PrPSc and opens many new avenues of research. Based on these findings, we plan to produce wt and mutant sequences of mouse, hamster, human and bovine prions from recombinant PrPs produced in E. coli. Next, we shall determine conditions under which different prion strains can be created by modifying the PrP amyloid polymerization process. We shall utilize some of the protocols that have been successful in creating different strains of yeast prions. The different strains will be characterized by incubation times, distribution of neuropathological changes, PrP deposition profiles in brain, measurements by the conformation-dependent immunoassay, conformational stability assays, as well as glycoform and protease-resistant fragment size analysis. We plan to attempt to identify PrPSc-like molecules within recPrP amyloid. The likelihood of success for this investigation will depend on the abundance of PrPSc in the amyloid fibrils. We plan to characterize the structural features of recPrP amyloids producing different prion strains. We also propose to develop an amyloid-screening assay for recombinant PrPs produced in E. coli. If this can be done, then we shall be able to identify an array of mutant prions, the infectivity of which will be assessed by bioassay in Tg mice.
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0.936 |
2006 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Anti-Prion Compounds/Cross the Blood Brain Barrier @ University of California San Francisco
[unreadable] DESCRIPTION (provided by applicant): We have developed a drug discovery pipeline targeting prion disease, and have been successful in identifying compounds that are effective in a cell-based model of prion disease. Our in vivo studies of these compounds have demonstrated that the efficacy of any anti-prion compound is dependent.on bioactivity and the ability to penetrate the blood-brain-barrier (BBB). We seek funding to expand our promising drug discovery pipeline to quantitate compound concentration in tissue and serum, following oral administration. This ADMET data will identify compounds that can achieve therapeutic concentrations within the CNS. To facilitate these in vivo experiments, we propose to undertake the multi-gram synthesis of any anti-prion compound that warrants in vivo study. Additionally, we have identified some important downstream applications of ADMET data for the design of CNS active anti-prion compounds. This proposed supplement [unreadable] is an important and necessary step in completing the research being conducted in Project 2 (Transgenic Mouse Models for Assessing Treatments of Prion Diseases), of this program project "Novel Therapeutics for Prion Diseases" (AG021601). Additionally, the data generated from this proposal is directly applicable to the ongoing research outlined in AG021601, Projects 3 (Combinatorial Synthesis of Quinacrine Analogs) and 4 (Understanding and Improving Small Molecule Inhibitors of Prion Replication). [unreadable] [unreadable]
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0.936 |
2006 — 2010 |
Prusiner, Stanley B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Preventing Human Prion Diseases - Inactivation of Prions @ University of California San Francisco
[unreadable] DESCRIPTION (provided by applicant): Prions are infectious proteins that cause neurodegenerative diseases in humans and animals. Prions exhibit unusual resistance to inactivation by chemical and physical methods that generally destroy infectious pathogens such as bacteria, fungi, and viruses. Normal hospital sterilization procedures do not inactivate prions, leading to the possibility of iatrogenic prion disease. The studies proposed here on the inactivation of a range of prion strains should increase our knowledge of prions substantially. Previous studies showed that prions become protease-sensitive after exposure to branched polyamines in the presence of a weak acid. These findings have been extended to the study of denaturing detergents, including sodium dodecyl sulfate (SDS) in acidic solutions. We have determined that treatment with acidic SDS combined with autoclaving reduces the infectivity of Syrian hamster Sc237 and human sCJD prions by a factor of >106. Prion inactivation will be studied using prion-infected brain homogenates and prion-coated wires, which serve as a model for surface contamination. Employing a battery of biophysical strategies, we plan to define the mechanism of prion inactivation by acidic SDS. These investigations may decipher the structural features of prions that make some strains extremely resistant and others quite labile to inactivation. Initially, we plan to study three strains of prions passaged in mice: RML and 301V, which originated from sheep with scrapie and cattle with BSE, respectively, plus a novel synthetic strain MoSP1, which is the most stable strain reported to date. We will undertake a systematic study of human sCJD prions to define the spectrum of stabilities, and produce novel human synthetic prion strains to extend the range of samples tested. Furthermore, we will attempt to identify noncorrosive denaturants that are more efficacious than acidic SDS, and adjuncts to acidic SDS treatment such as hydrogen peroxide, which may be as effective at lower temperatures. Finally, we plan to develop methods that will reduce the duration required for bioassays. Recent studies demonstrated that mice lacking the protein interleukin-10 are more susceptible to prion disease; we will breed these mice with our transgenic lines in the hope of reducing the incubation period. We also plan to develop an in-vitro assay for prion infectivity using neurospheres prepared from transgenic mice. The results from the studies proposed here have a very high likelihood of adding substantially to our basic knowledge of prion biology and leading to the development of effective procedures for inactivating prions. Effective protocols for inactivating prions will protect the general public as well as laboratory scientists who are investigating prions. Rarely does a research study have such immediate and important implications. [unreadable] [unreadable] [unreadable]
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0.936 |
2007 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core--Animal Facility @ University of California San Francisco
The function of the Animal Core is to produce and maintain all of the animals required for the scientific project for this and several other program project grants. The specific services of the Animal Core personnel include 1) Production of transgenic and knockout mice, 2) Production of large volumes of scrapie-infected hamsters and mouse brains for purification, 3) Performing experimental inoculations, neurologic scoring of animals, data collection, and tissue collection, 4) Providing transportation of animals and tissues between the laboratory and the animal facility, 5) Production of antibodies in mice and rabbits for experimental use, 6) Providing all animal care and veterinary care, 7) Providing cryopreservation of the various mouse lines. The Animal Core operates in two purpose-built facilities in the Hunters Point area of San Francisco, approximately 3 miles from the main UCSF campus. Its activities are directed by Dr. Prusiner and Dr. Pierre Lessard, a laboratory animal veterinarian. Building 830B is a new nine room transgenic mouse breeding facility that houses our large breeding colony. Building 830 houses all the experimental animals in 21 rooms, under BSL-2 and 3 biocontainment. It houses on average 10,000 mice, 400 hamsters, and 8 rabbits. The facility is inspected annually by USDA, and bi-annually by UCSF/IACUC.
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0.936 |
2007 — 2008 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Molecular Dissection of the Species Barrier For Prion Transmission @ University of California San Francisco
We propose to decipher the molecular basis of the species barrier by using transgenic (Tg) mice expressing chimeric human (Hu)/mouse (Mo) PrP genes. Earlier studies showed that mice expressing a chimeric PrP transgene designated MHu2M were susceptible to Hu prions. After systematically reverting some of the nine Hu residues to Mo at the N- and C-terminal ends of the "Hu2" insert, we identified Tg(MHu2M,S96N) mice with prolonged incubation times and Tg(MHu2M,M165V, E167Q) mice with abbreviated incubation times. The C-terminal residues 165 and 167 appear to be important in the binding of chimeric PrP to an auxiliary protein, provisionally designated protein X, that features in prion replication, while the N-terminal residues appear to bind to PrPSc in the inoculum. Our findings define a limited but feasible study of residues 96, 165 and 167 as well as the remaining six Hu residues in the Hu2 insert. Within the constraints posed by the need to construct Tg mice, we propose to introduce a substantial number of changes at the positions of the nine Hu residues in the Hu2 insert. Individual substitutions as well as combinations in the form of doublets and triplets will be made. We also plan to develop a cell culture system for the bioassay of chimeric PrP genes in ScN2a and ScGT1 cells by disrupting both copies of the MoPrP gene in these cells as described in Project 13. Such an assay would lift the constraints posed by the current need for Tg mice to test each chimeric PrP gene construct. From our studies of Tg mice, we should be able to determine the effects each residue in the Hu2 insert on incubation times for Hu prions and apply this knowledge to the development of rapid Tg mouse models for both Hu prion diseases and Chronic Wasting Disease (CWD) in cervids.
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0.936 |
2007 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Transgenic Mouse Models For Assessing Treatments of Prion Diseases @ University of California San Francisco
Prion diseases are neurodegenerative diseases of humans and animals, which are invariably fatal and lead to death within a year after onset of clinical symptoms. As with most other neurodegenerative diseases, no effective therapy is known. Potential therapeutics proposed for treatment of prion diseases include polysulfated anions, dextrans, and cyclic tetrapyrroles have been shown to increase survival time when given prior to prion infection in rodents, but not when given after infection has been initiated. Besides studies in rodents, mouse neuroblastoma (ScN2a) cells chronically infected with scrapie prions have been used to identify several candidate antiprion drugs but none of these drugs has been shown to be effective in halting prion diseases in either animals or humans. Based on recently published laboratory studies using cultured cells and a pilot clinical study in progress, we propose to perform a series of therapeutic-based experiments in transgenic (Tg) and non-Tg mice. Tg mice overexpressing (1) mouse (M) prion protein (PrP), (2) bovine (Bo) PrP or (3) a chimeric human-mouse PrP designated either MHu2M(M165V, E167Q) or MHu2M(M165V) will be used in these studies. Tg and non-Tg mice will be used to evaluate the toxicity of quinacrine isomers and a series of new compounds that are at least 10-fold more efficacious as determined in ScNa cells. After acute, short-term and longterm toxicity studies, treatment protocols for mice inoculated with prions will be initiated. Mice will begin to receive one of the quinacrine isomers or a new compound at point when 50%, 75% or 85% of the incubation time has elapsed. In most cases, the mice will be treated for 30 days. Two different strains of mouse or human prions will be used in these studies. Besides neuropathologic studies, samples of blood, muscle and brain will be taken for immunoassay determinations of PrP sc. The results of these studies are likely to provide important data leading toward the development of an effective therapeutic regimen for prion diseases in humans.
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0.936 |
2008 — 2017 |
Prusiner, Stanley B |
R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Towards Therapeutics For Neurodegenerative Diseases @ University of California, San Francisco
There is not a single FDA-approved drug that halts or even slows neurodegeneration in the CNS. Despite spending billions of dollars, neither industry nor academia has been able to develop a single drug that slows the progression of Alzheimer's (AD), Parkinson's (PD) and Creutzfeldt-Jakob (CJD) diseases as well as amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD). Despite this roadblock, there have been impressive advances in understanding the pathogenesis of all these disorders. A steady accumulation of experimental data argues that a different protein causes each neurodegenerative disease and these proteins acquire alternative structures that become self-propagating i.e., prions (Meyer-Luehmann et al., 2006; Clavaguera et al., 2009; Frost and Diamond, 2009; Olanow and Prusiner, 2009; Brundin et al., 2010; Cushman et al., 2010; Colby and Prusiner, In press). These findings are most gratifying since they provide an enlarging body of evidence in support of what now are regarded as prescient speculations (Prusiner, 1984, 2001). The understanding that all or most ofthe neurodegenerative diseases are caused by prions may give several new perspectives on the development of effective therapeutics. First, drugs that cure cultured cells infected with prions may not predict success in experimental animals or humans. Such is our experience with the antimalarial drug quinacrine that cured cultured cells but failed to extend the lives of either mice or humans (Collinge et al., 2009; Ghaemmaghami et al., 2009). Even when the level of quinacrine was increased almost 100-fold, in the brains of mice in which the P-gp transporter (Mdr1) has been knocked out, above the halfeffective concentration (EC50) in cultured cells, it failed to extend the incubation time (Ghaemmaghami et al., ^ 2009). We were able to gather convincing evidence showing that the most likely explanation for this therapeutic failure was a conformational change in PrP^*^ resulting in a drug-resistant prion strain. These results prompted us to develop a broad drug discovery program that has begun to identify lead compounds that are able to extend incubation times in mice (Ghaemmaghami et al., 2010; Gallardo-Godoy et al., 2011). Discovering drugs that can be used to treat neurodegeneration presents substantial challenges. With that said, we desperately need additional assays for measuring the efficacy of hits and leads to proceed through the process of drug discovery. More predictive assays of lead efficacy in cultured cells prior to studies in Tg rodents are critical to the development of effective therapeutics. Such cell assays could save an immense amount of time and resources. To improve our in vitro assessments of chemical libraries as well as groups of lead compounds, we plan to use a newly acquired Opera confocal microscope system for high throughput screening (HTS). This advanced system will allow us to measure both PrP^ and PrP^'^ in subcellular compartments. Despite successfully adapting ELISAs for measuring these PrP isoforms in HTS, we anticipate that the robust resolution of the Opera system will allow us to predict more accurately, from cell-based assays, which compounds will advance successfully through animal models. The adaption of bioluminescence to the in vivo readout of experimental scrapie and Alzheimer's disease in Tg mice (Tamgtiney et al., 2009a; Watts et al., 2011) has greatly facilitated our drug efficacy studies. Plans for improving these mouse models and extending the work into rats are described below in the research plan. Bigenic mice-on the Mdrl knockout background and with a luciferase reporter expressed under control of the Gfap promoter-are being bred for use in drug efficacy studies. Similar mice expressing luciferase are being bred, in which the mouse PrP gene has been knocked out and a chimeric human (Hu)/mouse (Mo) transgene is also expressed. Such mice become ill <80 days after inoculation with CJD(MM1) prions (Giles et al., 2010). In another set of studies, we have used RNAi libraries to identify auxiliary proteins that participate in the formation and replication of PrP¿*^ prions. These studies provide a route into the identification of new drug targets for antiprion drugs. More emphasis on these studies is planned since it seems likely that cocktails of drugs with different modes of action will be the most likely routes to successful treatment of neurodegenerative illnesses.
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0.936 |
2009 — 2011 |
Prusiner, Stanley B |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Turnover Rate of Prp Oligomers in the Brain @ University of California, San Francisco
This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The accumulation of protein aggregates is associated with many neurodegenerative diseases. The presence of these protein aggregates is direct evidence that normal protein metabolism is disrupted. Prions are a primary example of this effect, as they are infectious proteinaceous particles. Although strains of prions have been isolated which differ in incubation time and pathology, there is no nucleic acid component of the prion particles. Infection requires only that normal protein turnover is diverted to the formation of new aggregates. Understanding the propagation of prion disease and how these aggregates cause neurodegeneration requires that we investigate changes in protein turnover due to prion infection. Using nitrogen isotope labeling we will measure protein turnover on a proteome-wide scale to assess perturbations in global protein turnover during prion disease.
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0.936 |
2009 — 2013 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Investigations of Human Prion Disease @ University of California, San Francisco
To study the human prion diseases, transgenic (Tg) mice expressing chimeric prion proteins (PrP) comprised of mouse and human PrP sequences have been produced. By mutating the residues individually that differ between human and mouse PrP, we have produced Tg mice that are progressively more susceptible to human prions as evidenced by shorter and shorter incubation times. We propose to develop Tg mice even more sensitive human prions and use these as well as existing Tg mice for the detection of human prions. The bioassay data will be compared to measurements of both the protease-sensitive (s) PrPSc as well as protease-resistant (r) PrPSc. We shall also study the tissue distribution of prion infectivity as well as sPrPSc and rPrPSc in both people and animal models. Our studies may elucidate some of the factors that feature in the transmission of prions between humans and animals. Currently, the Tg mice that are most susceptible to human prions are those expressing a chimeric Mo/Hu PrP, which differs from MoPrP at just six residues. These Tg mice are susceptible to prions from patients who died of sporadic (s) Creutzfeldt- Jakob disease (CJD), in just 80 days. We plan to study systematically the effect of each of these six residues on susceptibility to prion infection, and determine how the expression level of the transgene impacts the incubation time to disease onset after inoculation. We shall also use guinea pigs as models for human prion diseases, having recently demonstrated that they uniquely reproduce the pathological features of variant (v) CJD. The large brain and blood volumes of the guinea pig makes them useful for CNS imaging studies as well as investigations of prions in blood and lymphoid tissues. Several studies suggest that the distribution of prions in brain and lymphoid tissues are quite different in sCJD versus vCJD patients. Whether guinea pigs will reproduce these differences in prion distribution remains to be established. While only four cases of vCJD occurred in the UK last year, the possibility that vCJD prions have contaminated the human blood supply cannot be ignored. Besides measuring prion titers by bioassays in Tg mice, we plan to measure the levels of both sPrPSc and rPrPSc. Recent advances in methods for measuring particularly sprpsc usjng tne amyloid seeding assay (ASA) are encouraging.
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0.936 |
2009 — 2013 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Animals @ University of California, San Francisco
In this application comprising four scientific projects and four-cores, we propose to continue our studies focused on neurodegeneration caused by human prion diseases, the most common of which is sporadic (s) CJD. Prions seem to be composed solely of PrPSc molecules, which are derived from a precursor PrPc by a poorly understood process. The studies described here are aimed at defining the structure of PrPSc, characterizing the interactions of small molecules with both PrPc and PrPSc and dissecting the molecular events governing the propagation of different human prion strains. In Project 1, we propose to study the interactions of polyoxometalates (POMs) with PrP,S c . The POM phosphotungstate anion [PW12O40] (PTA) binds specifically to PrPSc, but not to PrP . POMs are a large class of inorganic metal oxide clusters with rigid polyhedral structures displaying substantial variations in size, shape, and charge density. In Project 2, we propose to carry out fiber diffraction studies of the 55-residue MoPrP(89-143,P101L) peptide that causes inherited prion disease, a 20 mer wt PrP(106-126) peptide known to form amyloid fibrils as well as purified truncated (PrP 27-30) and full-length PrPSc, both of which are infectious in wild-type animals. In Project 3, we propose to study the prions causing sCJD. These studies are possible because our most sensitive Tg mouse line expressing chimeric human/mouse PrP succumbs to disease in ~80 days after inoculation with sCJD prions. Relatively rapid bioassays of human prions in these Tg mice make it practical to measure the titers of prions throughout brain as well as in peripheral organs and body fluids collected from dead sCJD patients. We also propose to develop guinea pig models of sCJD and variant (v) CJD. In Project 4, we propose to discover new ligands that bind to and stabilize human PrPc. We also plan to determine whether such ligands inhibit the conversion of HuPrPc into HuPrPSc. Using a virtual screening approach, large libraries of organic molecules will be docked against the known structure of HuPrPc. High-scoring compounds will be tested for binding biophysically, controlling for non-specific inhibition to which anti-amyloid inhibitors are prone. The ultimate goal of all the proposed studies is to define the molecular events that feature in the formation of human prions in order to develop therapeutics that cure the human prion diseases.
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0.936 |
2009 — 2013 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Administration @ University of California, San Francisco
The Administrative Core performs all administrative, accounting and clerical work related to the Program. Production of manuscripts and program reports, preparing financial reports , reviewing expenditures, monitoring budgets and coordinating appropriations. An executive committee composed of the senior investigators and ex-officio members is described in the Administratiive Core. We will perform annual reviews at which all of the work of the previous year is reviewed and discussed and critiques are prepared by outside reviewers. In addition, Dr. Prusiner will meet at least once per month with each of the Project Directors. A well-organized and efficient Adminitrative Core is vital for to Program described in this application.
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0.936 |
2009 — 2011 |
Prusiner, Stanley B |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Biochemical and Biophysical Characterization of Prion Protein 2d Crystals @ University of California, San Francisco
This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Prions cause transmissible, neurodegenerative diseases with invariably fatal progression. A prion is an infective particle consisting exclusively of the prion protein. Two forms are discriminated: the benign, cellular form PrPC, and the disease-causing "scrapie" isoform PrPSc, which is confirmationally rearranged. Since PrPC and PrPSc differ in structure, studies of each conformation are essential. Our apporach focuses on PrPSc. We developed a high-yield protein purification protocol. Our preparations contain prion protein 2D crystals, which are structurally analyzed with standard and low-dose cryo electron microscopy. However, to further the quality and purity of our samples, we need to identify contaminants/copurified proteins from our samples for their systematic removal. This is the task for mass spectrometry. On a collaborative basis, copurified proteins are to be identified by analysis of bands derived from Coomassie-stained polyacrylamide gels.
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0.936 |
2010 — 2011 |
Prusiner, Stanley B |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Dynamic Silac For the Study of Prion Propagation @ University of California, San Francisco
This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Prions are infectious protein aggregates which cause neurodegeneration and death in mammals. Cultured neuronal cells have been used in the study of prions. The study of prion propagation in these cell lines is complicated by an inability to differentiate prions supplied to initiate the infection from new cellular prions. Introducing an novel epitope into cellular prion protein has been used previously to identify prion origin. This technique is problematic because pertubations to protein structure may influence prion transmission. Labeled amino acids in the culture medium would be incorporated into newly synthesized prions. The isotopic label in combination with mass spectroscopy would allow us to identify prions produced in the cell culture. Collaboration with the UCSF Mass Spectrometry Facility would give us access to the instrumentation necessary to measure the extent of isotopic incorporation into the prions we have generated. We have had successful collaborations on similar projects. We envision that this project would use the protocols we have established previously.
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0.936 |
2011 — 2015 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Molecular Pathogenesis of Age-Dependent Cns Regeneration @ University of California, San Francisco
DESCRIPTION (provided by applicant): A steady accumulation of data argues that prion-like mechanisms feature in the pathogenesis of most or all age-dependent neurodegenerative diseases. For both the mammalian and fungal prions, we have succeeded in producing a variety of synthetic strains. The physical characteristics of the synthetic prion strains breed true upon passage and are predictive of infectivity: there is a clear relationship between the length of the incubation time and the structural stability for the mammalian prion strains. The ability to create different infectious conformations and introduce them into yeast makes the [PSI+] system uniquely well suited for studying how changes in the conformation of proteins determine their physiological impact. While elucidating the molecular mechanisms that specify the properties of prion strains is likely to create improved models for experimental AD, it will probably be insufficient in the quest for effective therapeutics. That being the case, we plan to study cultured neurons produced from fibroblasts using stem cell technology. A systematic screen of TFs identified a pool of 3 TFs that can induce mouse fibroblasts to differentiate into neurons. We plan to produce neurons from human fibroblasts obtained from carriers with APP or PrP gene mutations that cause familial AD or CJD, respectively. These neurons will be used in studies on the propagation of PrP and A? amyloids in cells and transgenic mice. Deciphering the language of prion strains will open many new avenues of investigation that are likely to reach far beyond the pathogenesis of neurodegeneration. We do not understand the rules of protein-based inheritance and have little appreciation of how prions function in the normal physiology of the cell as well as cause neurodegeneration. PUBLIC HEALTH RELEVANCE: More than 5 million Americans suffer from Alzheimer's disease (AD) caused by brain degeneration. Of all the neurodegenerative disorders, the prion diseases are arguably the best understood. We propose studies directed toward elucidating the pathogenic mechanisms responsible for neurodegeneration in AD and Creutzfeldt-Jakob disease. Able to create distinct prion strains from recombinant proteins produced in bacteria that infect mice or yeast, we plan to elucidate the structures of these synthetic prion strains and study their propagation in mouse and human neurons produced from fibroblasts by a remarkable new stem cell technology. Dissecting the processes that feature in the pathogenesis of prion-induced neurodegeneration may provide novel insights that direct development of effective therapeutics not only for the prion diseases but also for the most common neurodegenerative disorder, AD. REVIEW OF INDIVUDUAL COMPONENTS OF THE PROGRAM PROJECT CORE A: ADMINISTRATIVE CORE;Dr. Stanley Prusiner, Core Leader (CL) DESCRIPTION (provided by applicant): The Administrative Core performs all administrative, accounting and clerical work related to the Program, including production of manuscripts and program reports, preparing financial reports, reviewing expenditures, monitoring budgets and coordinating appropriations. An External Advisory Board will be appointed for a term of 5 years to review, annually, the program's progress. As well, an Executive Committee will be appointed to meet quarterly. A strong Administrative Core is vital to the success of this Program. PUBLIC HEALTH RELEVANCE: Not provided.
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0.936 |
2012 — 2015 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
De Novo Generation of Mammalian Prion Strains @ University of California, San Francisco
Prion diseases, such as Creutzfeldt-Jakob Disease (CJD), are degenerative disorders affecting the central nervous system. They are uniformly fatal and illicit death following a long period of steady neurodegeneration, dementia and motor disfunction. In prion diseases, an aberrantly folded conformer ofthe prion protein (PrPSc) propagates by catalyzing the post-translational misfolding of cellular PrP (PrPC). We have recently succeeded in recapitulating this phenomenon in vitro by converting purified, bacterially-expressed, recombinant PrP into infectious prions. The ability to create synthetic prions in cell-free systems has provided definitive proof of the protein-only hypothesis of prion propagation and has enabled us to begin deciphering the structural basis of protein-based infectivity. The specific aims of this project seek to utilize synthetic prions to gain insights into the mechanism of prion-induced neurodegeneration. We propose to create homogeneous and highly infectious preparations of synthetic prions. The synthetic prion preparations will be propagated in mouse bioassays and used for structural analyses. Disease phenotypes induced by synthetic prions will be assessed by two novel approaches for global analysis of proteome homeostasis. Effects on protein translation will be studied by ribosome profiling and changes in proteome turnover will be measured by comprehensive in vivo isotopic replacement. We will also attempt to infect induced neuronal (iN] cells with synthetic prions. These strategies will uncover relationships between the physical structure of prion strains and their biological properties and may provide valuable insights into the mechanism of neurodegeneration in prion diseases such as CJD. RELEVANCE (See instructions): The continuing aging ofthe American population has made the discovery of therapies against neurodegenerative disorders an important public health goal. This study will provide insight into the etiology of prion diseases such as Creutzfeldt-Jakob disease (CJD), and may illuminate the mechanism of neurodegeneration in more prevalent proteinopathies such as Alzheimer's disease and Parkinson's disease.
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0.936 |
2012 — 2015 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Administrative @ University of California, San Francisco
The Administrative Core performs all administrative, accounting and clerical work related to the Program, including production of manuscripts and program reports, preparing financial reports, reviewing expenditures, monitoring budgets and coordinating appropriations. An External Advisory Board will be appointed for a term of 5 years to review, annually, the program's progress. As well, an Executive Committee will be appointed to meet quarterly. A strong Administrative Core is vital to the success of this Program
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0.936 |
2012 — 2015 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Analysis of Neurodegenerative Mechanisms Using Human and Mous in Cells @ University of California, San Francisco
The recent identification of transcription factors (TFs) that can induce conversion of fibroblasts into pluripotent stem (IPS) cells makes it potentially possible to generate patient-specific neurons from fibroblasts. However, the neurons thus produced are difficult to obtain. The present project builds on preliminary results demonstrafing that it is possible to direcfiy convert adult fibroblasts or other adult non- neuronal cells into neurons, referred to as 'induced neuronal cells' (iN cells), without an iPS intermediate. The resulfing IN cells have all ofthe functional properties of neurons, including the ability to form funcfional synapses as assayed electrophysiology. Thus, the IN cell technology provides a novel, more facile approach to generating and studying human neurons, and opens up a new avenue to invesfigafing neurons from patients with various neurodegenerafive diseases. However, at this point the IN cell technology has only been employed to fibroblasts from young mice, and the effect of aging on iN cell generation and/or the generation of IN cells from human fibroblasts have not yet been established. Therefore, the present application proposes experiments in three specific aims that will systematically investigate the effect of aging on iN cell generafion (specific aim 1), develop the technology to generate IN cells from human fibroblasts (specific aim 2), and use the IN cell technology to study the properties of mouse and human neurons containing mutafions that are known to cause Alzheimer's disease (specific aim 3). These aims will be pursued by a combinafion of fissue culture experiments with cells cultured from mice and humans, cell biology, molecular biology, and electrophysiology. Together, the experiments proposed in this applicafion will develop a new avenue for studying human neurons and for invesfigafing the relationship of aging and neurodegenerafion, with the long term goal of using this technology for a better understanding of human aging and human disease. RELEVANCE (See instructions): This application will develop methods to generate neurons direcfiy from non-neuronal cells, allowing the production of neurons from skin fibroblasts of human patients. These methods will then be used to test the effects of aging and of Alzheimer's disease mutations on neuronal biology, with the long-term goal of establishing a better understanding of how aging and Alzheimer's disease alter neuronal function.
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0.936 |
2012 — 2015 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Animal @ University of California, San Francisco
instmctions): The function of the Animal Core is to produce and maintain all of the animals required for the scientific projects for this and several other program project grants. The specific services of the Animal Core personnel include 1) Providing the highest quality animal and veterinary care, 2) Breding of transgenic and knockout mice, 3) Performing experimental inoculafions, bioluminescent imaging, neurologic scoring of animals, and fissue collecfion, 4) Transportafion of animals and tissues between the laboratory and the animal facility, 5) Producfion of antibodies in mice for experimental use, 6) Performing microinjection of constructs to generate new transgenic lines, 7) Providing cryopreservation of the various mouse lines. The Animal core will perform bioassay experiments in support of all three projects in addifion to the Science core. Samples from the Animal core will be supplied to the Neuropathology core for analysis, and direcfiy to the Projects. The Animal Core operates in two purpose-built facilifies in the Hunters Point area of San Francisco, approximately 7 miles from the main UCSF campus. Its activifies are directed by Dr. Prusiner and Dr. Pierre Lessard, a laboratory animal veterinarian. Building 830B is a nine room transgenic mouse breeding facility that houses our large breeding colony. Building 830 houses all the experimental animals in 21 rooms, under BSL-2 and 3 biocontainment. It houses on average 20,000 mice, 250 hamsters, and 8 rabbits. The facility is inspected annually by USDA, and bi-annually by UCSF/IACUC. RELEVANCE (See instructions): Understanding the molecular pathogenesis of prion diseases is central to ultimately developing treatments for a range of neurodegenerative disorders. This Animal Core supports the projects by performing bioassay studies, and the maintence of animals for other core funcfion such as anifibody producfion.
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0.936 |
2012 — 2015 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Molecular Analysis of Yeast Prions @ University of California, San Francisco
Since Reed Wickner first proposed that otherwise obscure non-Mendelian states in S. cerevisiae, [URE3] and [PST], result from prion-like conversions of endogenous proteins, fungal prions in general and [PST] in particular, have proven to be uniquely powerful systems for exploring universal features of prion biology. A few ofthe major insights include direct demonstrafion ofthe protein only hypothesis of prion inheritance, elucidafion of molecular basis of prion strains as being enciphered in the conformation ofthe infectious protein, discovery of a proliferation of novel prion proteins and the suggestion that prion-based inheritance could be a ubiquitous mechanism for epigenetic control of protein function, and revelation that the host chaperone machinery plays a crifical role in catalyzing prion replication. As with all experiments, these findings also raise a host of fundamental questions such as the structural basis ofthe different strain conformations, how these different conformations are inherited and why do they differentially impact a cell's physiology, and perhaps most profoundly what is the biological role of prions. We now have the tools and the intellectual foundafion to begin to provide clear and concrete answers to these questions. These insights in turn should directly inform efforts to understand the principles of prion infectivity for the mammalian PrP protein and more generally to advance our understanding of how and why proteins misfold and how such misfolded forms impact a cell in both disease and non disease states. To accomplish this, we propose to focus on the following three areas: Define the structural basis of prion strain variants. Define how the primary structure of prion determines the spectrum of preferred strain variants Use ribosome profiling to define the physiological impact ofthe [PST] prion and how the prion strain conformation as well as the genetic background ofthe yeast modulate these effects. RELEVANCE (See instructions): Protein misfolding is a hallmark of a wide range of neurodegenerative disorders including prion diseases and far more common noninfectious disease such as Alzheimers and Parkinson.
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0.936 |
2012 — 2015 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Science @ University of California, San Francisco
The Science Core exists to provide reagents and services to the various projects within this Program project grant, and to the Neuropathology and Animal cores. The need for specific reagents and services are often common to multiple projects and cores, as they are for the projects on other Program project grants. The most efficient method of operation for providing reagents and services requiring complex, expensive equipment is therefore via a central core with a highly skilled staff. For this renewal ofthe Program project, the Science core will provide recombinant mouse PrP (with and without isotopic labels) for the generation of synthetic mammalian prions, and anti-PrP antibodies, for Project 1. Recombinant Sup35 will be produced for Project 2, in addition to constructs of Sup35 for development of transgenic mouse lines. The Science core will provide fluorescence activated cell sorting, and prepration of various A-beta aggregates (both synthetic and extracted from natural sources), for Project 3. Anti-PrP antibodies including recombinant Fabs will be supplied to the Neuropathogy core (Core C). The Animal core (Core D) will be supplied with isotopically labeled mouse diet in support of Project 1, and screening of all transgenic lines in suport of Projects 1, 2 and 3. Leadership for the Core will be provided by Dr. Kurt Giles, and Assistant Professor with over 15 years experience in neurodegenerative disease research, and by Dr Michael Silber, a Professor with over 30 years industrial and academic experience, who will act as co-director. Drs. Giles and Silber will be assisted by other scientists and research associates to provide the above functions.
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0.936 |
2012 — 2015 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Pathology @ University of California, San Francisco
Neuropathology Cores directed by Dr. DeArmond have been part of Stanley Prusiner PPGs for the past 25 years. The overall goal has been to test the hypothesis that emerged from Dr. DeArmond's early correlative kinefic studies of PrP [Sc] neurochemistry, PrP[Sc] immunohistochemistry, and neuropathological analysis during the course of prion diseases, which stated that formation and accumulation of PrP[Sc] in neurons are the cause ofthe clinically relevant neuropathological changes. All of our evidence today suggests that neurodegeneration in prion diseases progresses in stereotypical sequence of pathogenic events that underlies neurological dysfunction and degenerafion in prion disease. The sequence encompasses a progression of funcfional and neuropathological changes that begin with formafion and accumulafion abnormal protease resistant PrP¿*^ in neurons, proceeds rapidly to synaptic degeneration as PrP^^ accumulates in plasma membranes, and terminates in autophagic nerve cell death when PrP¿¿ accumulates in lysosomes and autophagosomes. The main theme of this PPG's Projects is prion strains. Project 1 proposes to create multiple synthetic prions and compare their physical characteristics with their pathologic phenotype in transgenic mice. In Project 2, yeast prions Sc4 and Sc37 will be mutated to test their ability to infect mammalian cells in vitro and in vivo. Project 3 proposes to use induced neuronal cells (iN cells) carrying mutations in genes associated with neurodegenerafion, such as APP, presenilin-1 and tau, and test whether the gene mutations predispose them for prion-like infecfion. The Neuropathology Core has all techniques and expertise to analyze the fissues provided to us. The resulfing pathologies may be novel.
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0.936 |
2015 — 2019 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core D: Animals @ University of California, San Francisco
Summary: Core D Animals The Animal Core is central to the Program Project, performing bioassay experiments for Projects 1, 2, 3 and 4, and supplying tissues to the Neuropathology core (Core C), and tail biopsies to the Science core (Core B) for screening. The Animal Core will provide the following services: the highest quality animal and veterinary care; breeding of transgenic and knockout mice; experimental inoculations, and neurologic scoring of animals; bioluminescence imaging; behavioral assessments; tissue collection and storage; transportation of animals and tissues between the animal facility and the laboratory; microinjection of DNA constructs to generate new transgenic lines; and cryopreservation of new transgenic mouse lines. The demonstration that inoculation of Aß and tau aggregates can initiate self-propagation in the appropriate mouse model is a clear analogy to the prion diseases. However a major limitation of the Aß and tau inoculation models is that the mice don't show any overt clinical signs, and the only way of determining the degree of disease progression is to remove the brain for biochemical and neuropathological analysis. Our pioneering work in the field of bioluminescence imaging (BLI) and near infrared (NIR) fluorescence imaing has allowed us to address this issue and monitor disease progression in vivo. We have developed transgenic mouse models incorporating a luciferase reporter, enabling monitoring by BLI, for propagation of the prion protein (PrP) and Aß, and NIR fluorescent ligands that bind to tau. These provide a suite of tools to determine the biological relevance of protein and peptide aggregates formed in the Projects of this grant.
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0.936 |
2015 — 2019 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core a: Administration @ University of California, San Francisco
Summary: Core A Administration The Administrative Core performs all administrative, accounting, editorial and clerical work related to the Program. Production of manuscripts and program reports, preparing financial reports, reviewing expenditures, monitoring budgets and coordinating appropriations will all fall under Core A. An executive committee composed of the senior investigators is described in the Administrative Core Research Plan. We will perform internal reviews as well as annual reviews at which all of the work of the previous year is reviewed and discussed and critiques are prepared by outside reviewers.
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0.936 |
2015 — 2019 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Project 1: Abeta, Tau and Prp Prions Causing Neurodegenerative Diseases @ University of California, San Francisco
Summary: Project 1 Prusiner There is increasing evidence from both cell culture and animal models that both the A? peptide and the tau protein become self-propagating in Alzheimer's disease (AD), initiating a cascade of neurodegeneration that spreads throughout the brain, indicating that A? and tau are prions. Thus, the molecular events underlying the pathogenesis of AD and tauopathies are reminiscent of Creutzfeldt-Jakob disease (CJD), in which the misfolding of the prion protein (PrP) into a self-templating isoform termed PrPSc initiates disease. However, to better understanding the structural and biochemical properties of self-propagating protein aggregates in AD requires the creation of cellular and animal bioassays capable of rapidly ascertaining the presence of these prions. We propose to study three different human prions composed of PrPSc, A? or tau, each of which cause age-dependent neurodegeneration. In these studies, we will take advantage of new paradigms that allows us to monitor disease progression and to diagnose neurodegeneration in living mice, in addition to novel cellular assays, greatly accelerating research efforts. In Aim 1 we will build on our recent observation that Tg(APP23) can propagate different A? strains. We propose to identify A? strain diversity from both natural and synthetic sources, both by bioassay and in cell culture. We also plan to generate new transgenic rat models. In Aim 2 we attempt to create a better models of sporadic and genetic human prion diseases by a combination of cell culture and transgenic mouse studies, based on studies demonstrating that bank vole PrP can faithfully propagate human prion strains. In Aim 3, we will investigate a cell model of intracellular tau aggregation to develop a cellular bioassay for tau prions using confocal fluorescence microscopy.
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0.936 |
2020 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core a: Administrative Core @ University of California, San Francisco
PROJECT SUMMARY The Administrative Core is critical in facilitating direction, communication and collaboration among all Projects and Cores to promote successful, synergistic outcomes for this Program Project grant. This will be accomplished via several avenues of approach. An Executive Committee composed of the Project Leads will meet quarterly to review progress, make scientific and administrative decisions, develop and engage quality control measures, and anticipate and resolve issues. We will hold an annual three-day Neurodegenerative Diseases Review Meeting at which work covered by this Program will be reviewed by a panel of experts from UCSF and our collaborators at the Uniformed Services University and Daiichi Sankyo Co. Ltd. The Administrative Core will provide financial oversight for the grant, including the preparation of budgets, processing and monitoring of appropriations, accounting and budgeting review, centralized purchasing and asset tracking, and other control point functions unique to a large and complex Program Project. Core A will also act as a central point of contact for the collection and dissemination of communication and information, and prepare manuscripts, reports and other documents related to the grant. In this way the Administrative Core will support and encourage productive interactions among the Program team and provide an organizational and administrative framework to optimize its opportunities for success.
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0.936 |
2020 |
Prusiner, Stanley B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Project 1: Pathobiology and Genetics of Prions Causing Alzheimer's Disease @ University of California, San Francisco
PROJECT SUMMARY The plaques and tangles of Alzheimer?s disease (AD) were first described over a century ago; we now know for the first time that the proteins of these deposits are composed of A? and tau prions, respectively. Being able to measure these two prions using rapid human cell bioassays in conjunction with transgenic rodent models, we are now able to attack many areas of AD research that were previously inaccessible. We propose to expand our cohort of 114 brain samples from patients with AD and other neurodegenerative diseases to more than 1,000 specimens from brain banks worldwide, including equal numbers of aged-matched and gender-matched controls from cognitively normal subjects. We will survey the molecular profile of prion strains using a battery of cell lines and proteomics and relate these findings to detailed neuropathology and whole genome sequencing of every sample. Along these lines, we will also procure brain samples from upwards of 500 individuals with Down syndrome (DS) because these people all develop cognitive impairment and AD neuropathology at very young ages and have both A? and tau prions in their brains as we describe in this proposal. We will investigate the influence of the APOE genotype status on the levels of A? and tau prions in the brains of AD and DS patients, and perform mechanistic studies using human neural cells and humanized rodents. Importantly, we plan to expand our studies on the induction of tau prions by A? prions using novel human cell and rodent models on a background of different genetic risk factors. To identify the most relevant species of A? prions for these experiments, we will create a broad repertoire of synthetic A? prions composed of different isoforms informed by inherited AD mutations or genetic mosaicism as well as results from our proteomics data in human tissue. Since studies of neural exosomes collected from patients with AD or DS show increased levels of A? and tau proteins compared to controls, we plan to determine if these proteins are prions. If that proves to be the case, then it may be possible to develop a more biologically relevant blood test for AD, which would have enormous utility in the disease diagnosis and progression as well as the discovery of effective therapeutics. Our collaborations described in this P01 will allow us to rapidly define the structural events that underlie the molecular pathogenesis of AD as studied in this Project, which would seem critical to developing more effective AD therapeutics.
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0.936 |