George A. Carlson - US grants

biomedical equipment purchase;

We are requesting funds to purchase equipment necessary for synthesizing specific peptides as needed by members of the listed user group. The equipment includes an Applied Biosystems automated solid phase peptide synthesizer and a preparative HPLC from Sota Chromatography, Inc., for purification of peptides generated by the Applied Biosystems synthesizer. The primary user group represents a sizeable subset of the faculties of the Molecular Biology and Biology Departments at Princeton and includes all or almost all of the research groups at Princeton that we have identified as likely to require this capability in the pursuit of their current research. The primary use of the proposed facility is to generate peptides to be used as immunogens to elicit antibodies that recognize a specific protein, or domain of a protein, of biological interest. This technology will be applied in the pursuit of a number of NIH funded research projects. These include regulation of plasmid propagation in yeast, genetic analysis of protein export, mutator genes in E. coli, regulation of development in Caulobacter crescentus, outer membrane structure/function analysis in E. coli, molecular analysis of Drosophila sex determination, adenovirus gene expression, and analysis of p53, a cellular tumor antigen. The availability of this instrument will promote the expeditious completion of these projects.

The role of the Beta/A4-amyloid precursor protein gene (APP) on Chr 21 in Alzheimer's disease(AD) is not clear. Two lines of evidence suggest a pivotal role for APP in AD. First, individuals with Down's syndrome (trisomy 21) exhibit accelerated development of the neuropathological features of AD, including abundant amyloid plaques. Overexpression of APP may be responsible for AD-like changes in trisomy 21, though there is no direct evidence that this is the case. Second, linkage between a missense mutation at APP residue 717 and early onset familial AD (FAD) recently has been demonstrated. Unfortunately, no mouse model exists that recapitulates the features of AD. Results from transgenic (Tg) muse experiments on the neurodegenerative diseases caused by prions suggest that the key to mimicking the pathological changes of AD in short lived rodents is obtaining dramatic overexpression of the proteins involved. One line of Tg mice with modest overexpression of APP695 encoded in a minigene construct is currently available. By use of a cosmid transgenic expression vector (cosTet), developed to express hybrid prion protein genes, additional Tg line overexpressing the 695, 751, and 770 isoforms of APP will be produced. By creating APP 'cassettes' for insertion into cosTet, FAD-linked mutations and the mutation linked with hereditary cerebral hemorrhage with amyloidosis-Dutch can be introduced into the expression vector. This will allow testing the hypothesis that these mutations cause disease. Because it is possible that APP overexpression is not a proximal cause of AD, experiments are included that have the potential to induce AD-like changes in Tg mice. Mouse neurological mutations that cause neurodegeneration will be combined with APP transgenes to test the hypothesis that progressive death of cells overexpressing APP initiates Beta-amyloid accumulation and plaque formation. Similar experiments using neurotropic retrovirus or scrapie prions as the neurodegenerative agents are also proposed. Tg mice overexpressing APP, either normal or mutant, also will provide a powerful tool for examining the influence of other genes that may be involved in Ad, and will allow direct testing of the role of the ratio of expression of the different isoforms of APP in Ad. The value of a mouse model for AD warrants this novel approach.

Construction of a new Biomedical Research Building for McLaughlin Research Institute will begin in the Spring of 1992 with an anticipated completion in the Summer of 1993. The space created by this structure will allow the hiring of an additional 4-6 research investigators. Because animal-related studies are, and will continue to be, an essential component of the scientific mission of this Institute, approximately 20% of the floor space and over 25% of construction expense will be devoted to a modern, centralized animal facility within this building. Current animal facilities are overcrowded, lacking several functional components essential in a modern animal facility and are not appropriately equipped to allow the maintenance of disease-free laboratory mice. The current building and operational deficiencies will be corrected by the construction of a new facility and the oversight of a veterinarian experienced in laboratory animal medicine. However, the current caging system does not provide a true cage-level barrier and the heavy cloth cage filters compromise the micro-environment of the cage and make daily observation of the animals difficult. In addition, the cage racks are immobile making frequent sanitation of the units difficult. The mobile, micro-isolator cage system requested on this proposal, coupled with the requested laminar-flow work stations, will provide an effective and proven cage barrier system when used with appropriate techniques. The number of cages requested will also allow a reduction in cage populations and an improvement in the micro-environment of the individual cage. A bedding dump station is also requested to minimize exposure of personnel to allergens and possible contaminants. With procurement of the cage system and completion of the new animal facility, the goal of a high quality program of animal care and use will be attained and accreditation from AAALAC will be sought.

The role of the Beta/A4-amyloid precursor protein gene (APP) on Chr 21 in Alzheimer's disease(AD) is not clear. Two lines of evidence suggest a pivotal role for APP in AD. First, individuals with Down's syndrome (trisomy 21) exhibit accelerated development of the neuropathological features of AD, including abundant amyloid plaques. Overexpression of APP may be responsible for AD-like changes in trisomy 21, though there is no direct evidence that this is the case. Second, linkage between a missense mutation at APP residue 717 and early onset familial AD (FAD) recently has been demonstrated. Unfortunately, no mouse model exists that recapitulates the features of AD. Results from transgenic (Tg) muse experiments on the neurodegenerative diseases caused by prions suggest that the key to mimicking the pathological changes of AD in short lived rodents is obtaining dramatic overexpression of the proteins involved. One line of Tg mice with modest overexpression of APP695 encoded in a minigene construct is currently available. By use of a cosmid transgenic expression vector (cosTet), developed to express hybrid prion protein genes, additional Tg line overexpressing the 695, 751, and 770 isoforms of APP will be produced. By creating APP 'cassettes' for insertion into cosTet, FAD-linked mutations and the mutation linked with hereditary cerebral hemorrhage with amyloidosis-Dutch can be introduced into the expression vector. This will allow testing the hypothesis that these mutations cause disease. Because it is possible that APP overexpression is not a proximal cause of AD, experiments are included that have the potential to induce AD-like changes in Tg mice. Mouse neurological mutations that cause neurodegeneration will be combined with APP transgenes to test the hypothesis that progressive death of cells overexpressing APP initiates Beta-amyloid accumulation and plaque formation. Similar experiments using neurotropic retrovirus or scrapie prions as the neurodegenerative agents are also proposed. Tg mice overexpressing APP, either normal or mutant, also will provide a powerful tool for examining the influence of other genes that may be involved in Ad, and will allow direct testing of the role of the ratio of expression of the different isoforms of APP in Ad. The value of a mouse model for AD warrants this novel approach.

Scrapie is a transmissible neurodegenerative disease that is caused by infectious pathogens called prions. The only macromolecule that has been identified in the scrapie prion is an abnormal isoform of the prion protein (PrPSc), which is encoded by a host gene. The PrP gene (Prn-p in mice) also controls scrapie susceptibility and the interval between inoculation and illness. Although many studies have failed to detect a scrapie-specific nucleic acid, these negative results do not exclude the possibility that the scrapie agent has a host-independent genome. The reported existence of numerous :microbiological strains" of scrapie prions argues for a host-independent informational component. However, some properties of prion isolates, previously attributed to a nucleic acid genome, can be explained as an epigenetic effect of the PrP allotype of the previous host. Taking advantage of Prn congenic mouse strains and transgenic mice developed for these studies, experiments will determine which properties of distinct prion isolates can be attributed to the primary structure of PrP. Cell lines from congenic mice will be established to provide a system to analyse scrapie isolate properties in vitro. The question of whether the PrP allotype and species barriers to scrapie transmission reflect host immune responses will be answered by incubation time studies in SCID (severe combined immunodeficiency) mice. Preferential conversion of PrP allotypes to PrPSc as an explanation for scrapie isolate properties on passage through different mouse strains will be addressed by producing allotype-specific antibodies or by direct protein sequencing. The primary structure of PrP cannot be invoked to account for the distinctive behavior of scrapie isolates in the same inbred mouse strain. Immunoaffinity enrichment for PrPSc and denaturation-renaturation experiments address the involvement of a second component of prions in determining scrapie isolate properties. Somatic mutation of the PrP gene may account for sporadic Creutzfeldt-Jakob disease in humans; targeting mutations through homologous recombination in neural cell lines followed by transplantation to syngeneic mice will examine this possibility. Construction of mice which harbor several insertions of multi-copy transgenes will permit determination of the influence of PrP expression of scrapie incubation period and examination of the possibility for spontaneous scrapie through somatic or germline mutation. An understanding of the mechanisms that lead to diverse symptomology, pathology, susceptibility and disease course within the single family of prion disorders will undoubtedly shed new light on degenerative disease in general.

Mutations in any one of three distinct genes can cause early-onset Alzheimer disease (AD) that is similar to the much more sporadic form that occurs most frequently in older individuals. An invariant feature of AD pathology is the accumulation of amyloid plaques in the brain, composed primarily of the highly amyloidogenic pathology is the accumulation of amyloid precursor protein (APP). Elevation of long-form Abeta peptide is observed in presymptomatic carriers of disease-linked mutation in APP, presenilin 1 (PS1) or presenilin 2 (PS2) suggesting that APP processing is central to the disease process. The identification of genes and mutations causing AD have made possible the development of transgenic mouse models that recapitulate aspects of AD, including development of amyloid plaques and behavioral abnormalities. More importantly, the biochemical pathways involved in D seem to be similar in mice and humans; for example, mutant but not wild-type,. PS1 transgenes cause elevation of Abeta1-42 derived from a human APP transgene or from the endogenous mouse APP gene. Genetically defined stocks and strains of mice offer a new avenue for exploration of AD. Age of onset has been shown to vary considerably in large families segregating a single FAD mutation suggesting the existence of genetic modifiers, and sporadic AD almost certainly has a genetic component. By exploiting natural variation among different inbred mouse strains, genetic control of susceptibility to effects of APP over-expression on survival, pathology, behavior and electrophysiology. The ultimate goal is to identify the genes involved. By transferring the Tg2576 transgene-array from the outbred stock that develops amyloid plaques onto different inbred backgrounds, it will be possible to determine the relationship of plaque load to disease phenotypes. Preliminary genetic analysis indicates the presence or absence of behavioral abnormalities in mice with amyloid plaques depends on genetic background rather than plaque prevalence. Testing the Tg2576 transgene array in hybrids with recombinant inbred strain panels will allow comparison of the genetics of complex behavioral and electrophysiological traits that require replicates measurements on genetically identical animals. These and similar experiments will help determine whether extracellular amyloid is essential for the cognitive decline and neuronal loss seen in AD. Finally, testing the effects of candidate genes, including apolipoprotein E and superoxide dismutase 1, on the phenotypes produced by APP over-expression provides a means to dissect disease pathways and refine mouse models for AD.

DESCRIPTION (provided by applicant) Prion diseases are neurodegenerative disorders of humans and animals that involve misfolding of prion protein (PrP). These diseases can present as genetic, infectious or sporadic illnesses and each type is often transmissible to experimental animals. Diseases caused by prions include scrapie, bovine spongiform encephalopathy (BSE), and Creutzfeldt-Jakob disease (CJD) in humans. All prion diseases involve changes in the conformation of PrP from its benign cellular isoform, PrPC, to a disease-specific isoform, PrPsc. PrPsc is probably the sole functional component of the infectious particle and different conformations of PrPsc can encipher properties of prion strains. Originally thought to be diseases caused by an exogenous slow virus, convincing evidence now indicates that prion diseases are disorders of protein conformation. The discovery of a new principle of infection resulted from research involving many disciplines focused on PrP. The four senior investigators on this Program Project application propose to extend their collaborative prion research efforts in a new direction. Although PrP is central to infectivity, disease susceptibility and pathogenesis, other molecules also are involved. Recent discoveries provide the first opportunity to study mechanisms of prion replication and pathogenesis by focusing on genes other than that encoding PrP. Treating scrapie incubation time as a quantitative trait allowed the identification chromosomal regions harboring prion incubation time modifier genes. These genes will be identified using positional cloning and positional candidate approaches, aided by new technologies in DNA analysis, expression microarrays and BAC engineering for production of transgenic mice. Additional prion modifier genes will be identified by sampling natural polymorphisms in mouse strains using QTL analysis and by applying chemical mutagenesis to screen for novel prion incubation time genes. The first gene encoding a PrP-related protein, Dpl, was discovered downstream from the PrP gene. Although PrP and DpI exhibit only 25% sequence identity, they share a conserved three-helix bundle structure. This provides a unique opportunity to learn more about prion replication and disease pathogenesis through creation of chimeric PrP:Dpl molecules. DpI misexpression in brain causes cerebellar neurodegeneration that can be prevented by PrP expression, suggesting involvement of similar pathways which will be interrogated with cDNA arrays and by identification of modifier genes. In addition to providing a new perspective on prion disease, these studies may ultimately reveal pathways common to a variety of CNS degenerative disorders.

DESCRIPTION (provided by applicant): Temperature gradient capillary electrophoresis (TGCE) using the SpectuMedix Reveal-2410 Mutation Discovery System will accelerate identification of the DNA sequence underlying biological phenotypes and developmental processes. By combining temperature gradient electrophoresis with capillary electrophoresis it is possible to rapidly identify DNA fragments with mismatches in nucleotide sequence based on the melting temperature. By running sufficiently broad temperature gradients, mismatches in multiple fragments differing in size and nucleic acid sequence can be detected at efficiencies above 95% in a single run. This is a major advantage over other approaches to identify functional nucleotide sequence changes. Rather than sequencing an entire candidate gene, the Mutation Discovery System prioritizes the exon or region of DNA with mismatch between mutant and wild-type for nucleotide sequencing. The Reveal-2410 can then be used as a standard nucleotide sequencer to identify the nucleotide changes. Increased nucleotide sequencing is also needed due to expanded use of phage-based E. coli homologous recombination for rapid modification of large segments of genomic DNA for generation of constructs for knockout and transgenic mice. The instrument requested will benefit NIH funded research programs in the genetic control of rnyelination by Schwann cells, in the functional genomics of susceptibility to prion diseases, in the role of molecular motors in hearing, and in the developmental biology of the auditory system and congenital deafness. In addition to the demonstrated ability of this technology to reveal single base changes in fragments between 200 to 500 bp, such as isogenic mutations induced by ENU, novel uses, such ranking candidate genes underlying QTL, are likely to prove fruitful. The Spectrumedix Reveal 2410 will not only accelerate current research at McLaughlin Research Institute, but help in recruiting new biomedical researchers in the field of neurogenetics.

[unreadable] DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a progressive neurodegenerative disease. It is prevalent among the elderly, affecting approximately 4 million Americans. Alzheimer's is a heterogeneous disease involving a number of components, including genetics. Although the accumulation of beta-amyloid peptide (AB) has been associated with both familial and sporadic forms of Alzheimer's, our current understanding of the role of AB and the mechanisms leading to the decline of cognitive function and neuronal loss are speculative. Transgenic mouse lines that over express AB peptides develop amyloid plaques and show age-related memory deficits have been created. Unfortunately, these mouse models for AD do not result in progressive neuronal loss as seen in humans. Whether this is due to species differences between mouse and human neurons can now be addressed directly by examining the fate of human neural cells transplanted into these AD mouse models. The ability to identify, isolate and expand human neural stem cells provides the opportunity to generate well-characterized cells for transplantation. In vivo properties of these cells have been extensively tested in the experimental NOD-Scid xenogeneic transplant mouse system. The striking features of the human CNS-SC is their capacity to engraft, migrate within the brain, and phenotypically differentiate into the three major cell types, neurons, astrocytes, and oligodendrocytes. Here we propose to examine the fate of human CNS-SC derived neural cells in the mouse models that recapitulate features of AD to assess the utility of neural cell transplants in the treatment of AD. Moreover, one can envision that these stem cells by their very biological property could produce and replace lost or dysfunctional neurons. The objectives of this grant are to test human neural stern cells as candidate therapeutics for the treatment of Alzheimer's disease. [unreadable] [unreadable] [unreadable]

[unreadable] Description (provided by applicant): The goals of this project are to dissect the biochemical networks that impinge on prion replication and to learn how prion replication causes neuronal dysfunction. The identification and characterization of genes in addition to Prnp that modify prion replication and susceptibility to prion disease has proven exceptionally difficult due to the need for incubation time studies in mice. Even the mechanisms by which alternative alleles of Prnp determine scrapie incubation time are unresolved. CNS stem cell neurosphere cultures can be produced from any strain or stock of mice, as well as from other species, and can be infected with prions. Neurosphere cultures provide a new approach to study prion biology and will be developed as a sensitive bioassay. Efficiency of infection, spread from cell to cell, and rate of prion replication can be discriminated in neurosphere cultures. Conformation dependent epitopes allow identification of individual cells with intracellular PrPSc; quantitative assays for infection, spread, and rate of replication will be refined. These parameters will be compared in prion strain-mouse strain combinations that vary in prion susceptibility or incubation time. Neurospheres that recapitulate the susceptibility of the mice of origin will form the substrates for dissection of the mechanisms and genes that are involved. Based on the hypothesis that alternative alleles of many quantitative trait loci reflect either differences in level or expression or different affinities for interacting molecules, RNAi will be used to evaluate effects of candidate genes on infection and production of PrPSc. The ability to produce neurosphere lines from any strain of mice permits testing modifiers using the same genetic background on which they were detected. CNS stem cells can be induced to differentiate along several pathways. The cell types produced under specific conditions from infected and non-infected neurosphere cultures will be compared, as will cell survival. Brain homogenates from mice infected with the RML prion strain diluted 10-8 can infect neurospheres from transgenic mice overexpressing PrP, and the limits of sensitivity have not yet been reached. Shortening the assay time from weeks to days will employ antibody epitopes present on PrP of the neurospheres, but not in PrPSc in the inoculum. This genetically tractable cell culture system has the potential to revolutionize prion research. [unreadable] [unreadable] [unreadable]

The administrative support performed in this core is directed to ensuring efficient interactions among the investigators at the three institutions that are involved in the project. In addition to handling subcontracts and serving as a liaison between the Institute's financial office and the other institutions involved, dedicated support is required to ensure coordination of shipments and scheduling essential for the success of the project. The Principal Investigator will have overall and final responsibility for the scientific and financial management of the program and will act in consultation with the project leaders. An Executive Committee involving all Project Leaders and an outside advisor will be formed. The administrative core will provide a structure that provides for regular input from all component programs. The Administrative Core performs all administrative and some secretarial work associated with the program. Included are monitoring timely payments to subgrantees and coordinating activities between the three institutions. An Executive Committee involving all Project Leaders and an outside advisor will be formed. Since this project requires extensive and timely interactions among the different project leaders and their staffs, coordination of shipments of materials, animals and data require administrative attention for smooth functioning.

DESCRIPTION (provided by applicant): Neurodegenerative protein misfolding diseases, the most common of which is Alzheimer's disease (AD), exact devastating personal and economic tolls on society. CNS stem cell (CNS-SC) transplantation has been touted, justifiably, as a promising approach for treatment of Alzheimer's disease (AD) and other degenerative brain diseases either by replacing lost cells or as a vehicle for delivery of therapeutic agents. CNS-SC transplantation also may offer new approaches for dissecting disease processes and mechanisms. The broad aim of this pilot project is to assess and refine neurosphere transplantation as a tool to dissect pathogenic mechanisms and spread of tau pathology. Two paradigms will be explored: 1) transplantation of CNS-SC containing neurospheres from mice that do not develop disease into disease models, and 2) transplantation of neurospheres from disease models into mice that do not develop disease. The substrate for these pilot studies is the well-characterized rTg(tauP301L)4510 model for the tauopathy frontotemporal dementia (FTD). AD is characterized by extracellular Ass amyloid plaques and intracellular neurofibrillarly tangles (NFTs) composed of aggregated tau protein. Of the two, NFTs are more tightly associated with cognitive decline, neuron loss, and dementia than are plaques, arguing for the relevance of the FTD model for AD as well as other tauopathies. Evidence suggests that soluble extracellular forms of tau, rather than intracellular neurofibrillary tangles (NFTs), may be the triggers of neuronal dysfunction and cell death. Experiments will compare transplants of neurospheres from eGFP-expressing tau null and mouse tau-expressing mice into newborn rTg(tauP301) and rTg(tauwt) mice that express comparable levels of human mutant or wild type tau, and track cell survival, differentiation into neurons, and synapse formation by the progeny of the transplanted cells. In addition to addressing the question of whether endogenous tau expression is required for deleterious effects of extracellular tau aggregates, replacement of lost host neurons will be assessed. The project will address the questions of whether neurons from rTg(tauP301L) neurospheres develop tauopathy and whether spread of tauopathy is influenced by mismatching of tau primary structures in the transplant and the host. Refined methods and tools will be developed to facilitate application of stem cell transplantation to understanding disease mechanisms; these should prove valuable beyond this specific project.

SUMMARY Tauopathy i n A D a n d F T D is associated with diverse disease syndromes, and e s p e c i a l l y f o r F T D , there is variability in phenotype even w i t h i n f a m i l i e s , a n d between patients with the same mutation. Currently, little is known about why abnormal tau can give rise to such varied phenotypes, but recent data have suggested that subtypes of tau (strains) might underlie this diversity. Our working hypothesis is that p h e n o t y p i c d i v e r s i t y i n t h e t a u o p a t h i e s r e f l e c t s t h e c o n t r i b u t i o n o f differentstrainsthatimpactdifferentbrainregions.Ourproposalaimstousebiochemicaland structural analysis techniques to define strains and sophisticated longitudinal imaging to study how and where in the cell strain variants of tau are propagated, and through the use of biosensors, study the effect of different tau strains on cellular processes. Strains will then be inoculated into mice to study pathology morphology and distribution, as well as functional impact. Together, the aims of this proposal will benefit patients w i t h A D a n d F T D by fostering a better understanding of tauopathy, and providing valid cell and animal models for the testing of therapeutics.

PROJECT SUMMARY At present, there is no treatment that prevents or even slows the progression of Alzheimer's disease (AD), which is the most prevalent neurodegenerative disorder. The Animal Core (Core C) will provide normal and genetically modified mice or rats and will conduct the experiments described in Projects 1, 2, and 3. Rodent models that express human proteins involved in neurodegenerative disease proved key in the discovery of misfolded forms of normal proteins, called prions, as new principles of disease. AD involves two prions, tau and Abeta. Cellular assays allow measurement of human tau and Abeta prions in culture, but transfer of disease from humans to mice will provide essential validation. Inoculation of brain homogenates into specific regions of the rodent brain and subsequent diagnosis of disease requires exceptionally skilled animal technicians who also assess disease onset by evaluating clinical signs and by bioluminescence as a marker of disease-induced gliosis in transgenic models specifically bred for this purpose (Projects 1 and 2). The stability of tau and Abeta prion strains or conformations also will be tested by repeated passage in mice (Project 2). Exceptionally high resolution structures (Project 3) can be determined by cryo-electron microscopy using material provided by Core C. The experiments executed in Core C ultimately will provide models to test therapies that halt disease progression.